Passive immunization treatment of Alzheimer&#39;s disease

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with amyloid deposits of Abeta in the brain of a patient. Such methods entail administering agents that induce a beneficial immunogenic response against the amyloid deposit. The methods are useful for prophylactic and therapeutic treatment of Alzheimer&#39;s disease. Preferred agents including N-terminal fragments of Abeta and antibodies binding to the same.

TECHNICAL FIELD

The invention resides in the technical fields of immunology andmedicine.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16, 403-409 (1993); Hardy et al.,WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994);Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523(1995). Broadly speaking, the disease falls into two categories: lateonset, which occurs in old age (65+years) and early onset, whichdevelops well before the senile period, i.e., between 35 and 60 years.In both types of disease, the pathology is the sarre but theabnormalities tend to be more severe and widespread in cases beginningat an earlier age. The disease is characterized by at least two types oflesions in the brain, senile plaques and neurofibrillary tangles. Senileplaques are areas of disorganized neuropil up to 150 μm across withextracellular amyloid deposits at the center visible by microscopicanalysis of sections of brain tissue. Neurofibrillary tangles areintracellular deposits of microtubule associated tau protein consistingof two filaments twisted about each other in pairs.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is an internal fragment of 39-43 aminoacids of a precursor protein termed amyloid precursor protein (APP).Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature 349,704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353, 844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254, 97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1, 345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20, 154 (1997)). These observations indicate that Aβ, andparticularly its long form, is a causative element in Alzheimer'sdisease.

McMichael, EP 526,511, proposes administration of homeopathic dosages(less than or equal to 10⁻² mg/day) of Aβ to patients withpreestablished AD. In a typical human with about 5 liters of plasma,even the upper limit of this dosage would be expected to generate aconcentration of no more than 2 pg/ml. The normal concentration of Aβ inhuman plasma is typically in the range of 50-200 pg/ml (Seubert et al.,Nature 359, 325-327 (1992)). Because EP 526,511's proposed dosage wouldbarely alter the level of endogenous circulating Aβ and because EP526,511 does not recommend use of an adjuvant, as an immunostimulant, itseems implausible that any therapeutic benefit would result.

By contrast, the present invention is directed inter alia to treatmentof Alzheimer's and other anyloidogenic diseases by administration offragments of Aβ, or antibody to certain epitopes within Aβ to a patientunder conditions that generate a beneficial immune response in thepatient. The invention thus fulfills a longstanding need for therapeuticregimes for preventing or ameliorating the neuropathology and, in somepatients, the cognitive impairment associated with Alzheimer's disease.

This application is related to International Application No.PCT/US00/14810 filed May 26, 2000, Publication No. WO 00/72880; U.S.application Ser. No. 322,289, filed May 28 1999; PCT/US98/25386, filedNov. 30, 1998, Publication No. WO 99/27944; U.S. application Ser. No.09/201,430, filed Nov. 30, 1998; U.S. Application No. 60/067,740, filedDec. 2, 1997; and, U.S. Application No., filed Apr. 7, 1998; each ofwhich is incorporated by reference in its entirety for all purposes.

SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides methods of preventing or treatinga disease associated with amyloid deposits of Aβ in the brain of apatient. Such diseases include Alzheimer's disease, Down's syndrome andcognitive impairment. The latter can occur with or without othercharacteristics of an amyloidogenic disease. Some methods of theinvention entail administering an effective dosage of an antibody thatspecifically binds to a component of an amyloid deposit to the patient.Such methods are particularly useful for preventing or treatingAlzheimer's disease in human patients. Some methods entail administeringan effective dosage of an antibody that binds to Aβ. Some methods entailadministering an effective dosage of an antibody that specifically bindsto an epitope within residues 1-10 of Aβ. In some methods, the antibodyspecifically binds to an epitope within residues 1-6 of Aβ. In somemethods, the antibody specifically binds to an epitope within residues1-5 of Aβ. In some methods, the antibody specifically binds to anepitope within residues 1-7 of Aβ. In some methods, the antibodyspecifically binds to an epitope within residues 3-7 of Aβ. In somemethods, the antibody specifically binds to an epitope within residues1-3 of Aβ. In some methods, the antibody specifically binds to anepitope within residues 1-4 of Aβ. In some methods, the antibody bindsto an epitope comprising a free N-terminal residue of Aβ. In somemethods, the antibody binds to an epitope within residues of 1-10 of Aβwherein residue 1 and/or residue 7 of Aβ is aspartic acid. In somemethods, the antibody specifically binds to Aβ peptide without bindingto full-length amyloid precursor protein (APP). In some methods, theisotype of the antibody is human IgG1.

In some methods, the antibody binds to an amyloid deposit in the patientand induces a clearing response against the amyloid deposit. Forexample, such a clearing response can be effected by Fc receptormediated phagocytosis.

The methods can be used on both asymptomatic patients and thosecurrently showing symptoms of disease. The antibody used in such methodscan be a human, humanized, chimeric or nonhuman antibody and can bemonoclonal or polyclonal. In some methods, the antibody is prepared froma human immunized with Aβ peptide, which human can be the patient to betreated with antibody.

In some methods, the antibody is administered with a pharmaceuticalcarrier as a pharmaceutical composition. In some methods, antibody isadministered at a dosage of 0.0001 to 100 mg/kg, preferably, at least 1mg/kg body weight antibody. In some methods, the antibody isadministered in multiple dosages over a prolonged period, for example,of at least six months. In some methods, the antibody is administered asa sustained release composition. The antibody can be administered, forexample, intraperitoneally, orally, subcutaneously, intracranially,intramuscularly, topically, intranasally or intravenously.

In some methods, the antibody is administered by administering apolynucleotide encoding at least one antibody chain to the patient. Thepolynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody. The polynucleotide is expressed to produce the heavyand light chains in the patient. In some methods, the patient ismonitored for level of administered antibody in the blood of thepatient.

In another aspect, the invention provides methods of preventing ortreating a disease associated with amyloid deposits of Aβ in the brainof patient. For example, the methods can be used to treat Alzheimer'sdisease or Down's syndrome or cognitive impairment. Such methods entailadministering fragments of Aβ or analogs thereof eliciting animmunogenic response against certain epitopes within Aβ. Some methodsentail administering to a patient an effective dosage of a polypeptidecomprising an N-terminal segment of at least residues 1-5 of Aβ, thefirst residue of Aβ being the N-terminal residue of the polypeptide,wherein the polypeptide is free of a C-terminal segment of Aβ. Somemethods entail administering to a patient an effective dosage of apolypeptide comprising an N-terminal segment of Aβ, the segmentbeginning at residue 1-3 of Aβ and ending at residues 7-11 of Aβ. Somemethods entail administering to a patient an effective dosage of anagent that induces an immunogenic response against an N-terminal segmentof Aβ, the segment beginning at residue 1-3 of Aβ and ending at residues7-11 of Aβ without inducing an immunogenic response against an epitopewithin residues 12-43 of Aβ43.

In some of the above methods, the N-terminal segment of Aβ is linked atits C-terminus to a heterologous polypeptide. In some of the abovemethods, the N-terminal segment of Aβ is linked at its N-terminus to aheterologous polypeptide. In some of the above methods, the N-terminalsegment of Aβ is linked at its N and C termini to first and secondheterologous polypeptides. In some of the above methods, the N-terminalsegment of Aβ is linked at its N terminus to a heterologous polypeptide,and at its C-terminus to at least one additional copy of the N-terminalsegment. In some of the above methods, the heterologous polypeptide andthereby a B-cell response against the N-terminal segment. In some of theabove methods, the polypeptide further comprises at least one additionalcopy of the N-terminal segment. In some of the above methods, thepolypeptide comprises from N-terminus to C-terminus, the N-terminalsegment of Aβ, a plurality of additional copies of the N-terminalsegment, and the heterologous amino acid segment. In some of the abovemethods, the N-terminal segment consists of Aβ1-7. In some of the abovemethods, the N-terminal segment consists of Aβ3-7.

In some methods, the fragment is free of at least the 5 C-terminal aminoacids in Aβ43. In some methods, the fragment comprises up to 10contiguous amino acids from Aβ. Fragments are typically administered atgreater than 10 micrograms per dose per patient.

In some methods, the fragment is administered with an adjuvant thatenhances the immune response to the Aβ peptide. The adjuvant andfragment can be administered in either order or together as acomposition. The adjuvant can be, for example, aluminum hydroxide,aluminum phosphate, MPL™, QS-21 (Stimulon™) or incomplete Freund'sadjuvant.

The invention further provides pharmaceutical compositions comprisingfragments of Aβ or other agents eliciting immunogenic response to thesame epitopes of Aβ, such as described above, and an adjuvant. Theinvention also provides pharmaceutical compositions comprising any ofthe antibodies described above and a pharmaceutically acceptablecarrier.

In another aspect, the invention provides methods of screening anantibody for activity in treating a disease associated with deposits ofAβ in the brain of a patient (e.g., Alzheimer's disease). Such methodsentail contacting the antibody with a polypeptide comprising at leastfive contiguous amino acids of an N-terminal segment of Aβ beginning ata residue between 1 and 3 of Aβ, the polypeptide being free of aC-terminal segment of Aβ. One then determines whether the antibodyspecifically binds to the polypeptide, specific binding providing anindication that the antibody has activity in treating the disease.

In another aspect, the invention provides methods of screening anantibody for activity in clearing an antigen-associated biologicalentity. Such methods entail combining the antigen-associated biologicalentity and the antibody and phagocytic cells bearing Fc receptors in amedium. The amount of the antigen-associated biological entity remainingin the medium is then monitored. A reduction in the amount of theantigen-associated biological entity indicates the antibody has clearingactivity against the antigen-associated biological entity. The antigencan be provided as a tissue sample or in isolated form. For example, theantigen can be provided as a tissue sample from the brain of anAlzheimer's disease patient or a mammal animal having Alzheimer'spathology. Other tissue samples against which antibodies can be testedfor clearing activity include cancerous tissue samples, virally infectedtissue samples, tissue samples comprising inflammatory cells,nonmalignant abnormal cell growths, or tissue samples comprising anabnormal extracellular matrix.

In another aspect, the invention provides methods of detecting anamyloid deposit in a patient. Such methods entail administering to thepatient an antibody that specifically binds to an epitope within aminoacids 1-10 of Aβ, and detecting presence of the antibody in the brain ofthe patient. In some methods, the antibody binds to an epitope withinresidues 4-10 of Aβ. In some methods, the antibody is labelled with aparamagnetic label and detected by nuclear magnetic resonancetomography.

The invention further provides diagnostic kits suitable for use in theabove methods. Such a kit comprises an antibody that specifically bindsto an epitope with residues 1-10 of Aβ. Some kits bear a labeldescribing use of the antibody for in vivo diagnosis or monitoring ofAlzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Antibody titer after injection of transgenic mice with Aβ1-42.

FIG. 2: Amyloid burden in the hippocampus. The percentage of the area ofthe hippocampal region occupied by amyloid plaques, defined byreactivity with the Aβ-specific monoclonal antibody 3D6, was determinedby computer-assisted quantitative image analysis of immunoreacted brainsections. The values for individual mice are shown sorted by treatmentgroup. The horizontal line for each grouping indicates the median valueof the distribution.

FIG. 3: Neuritic dystrophy in the hippocampus. The percentage of thearea of the hippocampal region occupied by dystrophic neurites, definedby their reactivity with the human APP-specific monoclonal 8E5, wasdetermined by quantitative computer-assisted image analysis ofimmunoreacted brain sections. The values for individual mice are shownfor the AN1792-treated group and the PBS-treated control group. Thehorizontal line for each grouping indicates the median value of thedistribution.

FIG. 4: Astrocytosis in the retrosplenial cortex. The percentage of thearea of the cortical region occupied by glial fibrillary acidic protein(GFAP)-positive astrocytes was determined by quantitativecomputer-assisted image analysis of immunoreacted brain sections. Thevalues for individual mice are shown sorted by treatment group andmedian group values are indicated by horizontal lines.

FIG. 5: Geometric mean antibody titers to Aβ1-42 following immunizationwith a range of eight doses of AN1792 containing 0.14, 0.4, 1.2, 3.7,11, 33, 100, or 300 μg.

FIG. 6: Kinetics of antibody response to AN1792 immunization. Titers areexpressed as geometric means of values for the 6 animals in each group.

FIG. 7: Quantitative image analysis of the cortical amyloid burden inPBS- and AN1792-treated mice.

FIG. 8: Quantitative image analysis of the neuritic plaque burden inPBS- and AN1792-treated mice.

FIG. 9: Quantitative image analysis of the percent of the retrosplenialcortex occupied by astrocytosis in PBS- and AN1792-treated mice.

FIG. 10: Lymphocyte Proliferation Assay on spleen cells fromAN1792-treated (FIG. 10A) or PBS-treated (FIG. 10B).

FIG. 11: Total Aβ levels in the cortex. A scatterplot of individual Aβprofiles in mice immunized with Aβ or APP derivatives combined withFreund' adjuvant.

FIG. 12: Amyloid burden in the cortex was determined by quantitativeimage analysis of immunoreacted brain sections for mice immunized withthe Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; the full length Aβaggregates AN1792 (Aβ1-42) and AN1528 (Aβ1-40) and the PBS-treatedcontrol group.

FIG. 13: Geometric mean titers of Aβ-specific antibody for groups ofmice immunized with Aβ or APP derivatives combined with Freund'sadjuvant.

FIG. 14: Geometric mean titers of Aβ-specific antibody for groups ofguinea pigs immunized with AN1792, or a palmitoylated derivativethereof, combined with various adjuvants.

FIGS. 15 A—E: Aβ levels in the cortex of 12-month old PDAPP mice treatedwith AN1792 or AN1528 in combination with different adjuvants. The Aβlevel for individual mice in each treatment group, and the median, mean,and p values for each treatment group are shown.

FIG. 15A: The values for mice in the PBS-treated control group and theuntreated control group.

FIG. 15B: The values for mice in the AN1528/alum andAN1528/MPL-treatment groups.

FIG. 15C: The values for mice in the AN1528/QS21 and AN1792/Freund'sadjuvant treatment groups.

FIG. 15D: The values for mice in the AN1792/Thimerosol and AN1792/alumtreatment groups.

FIG. 15E: The values for mice in the AN1792/MPL and AN1792/QS21treatment groups.

FIG. 16: Mean titer of mice treated with polyclonal antibody to Aβ.

FIG. 17: Mean titer of mice treated with monoclonal antibody 10D5 to Aβ.

FIG. 18: Mean titer of mice treated with monoclonal antibody 2F12 to Aβ.

FIG. 19: Epitope Map: Restricted N-terminal Response. Day 175 serum fromcynomolgus monkeys was tested by ELISA against a series of 10-meroverlapping peptides (SEQ ID NOS:1-41) covering the complete AN1792sequence. Animal number F10920M shows a representative N-terminalrestricted response to the peptide DAEFRHDSGY (SEQ ID NO:9) which coversamino acids 1-10 of the AN1792 peptide which was used as immunizingantigen.

FIG. 20: Epitope Map: Non-restricted N-terminal response. Day 175 serumfrom cynomolgus monkeys was tested by ELISA against a series of 10-meroverlapping peptides (SEQ ID NOS:1-41) covering the complete AN1792sequence. Animal number F10975F shows a representative non-restrictedN-terminal response. Reactivity is seen against the two peptidesN-terminal and one peptide C-terminal to the peptide DAEFRHDSGY (SEQ IDNO:9) which covers amino acids 1-10 of the AN1792 peptide.

DEFINITIONS

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAβ or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). Typically, default program parameterscan be used to perform the sequence comparison, although customizedparameters can also be used. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff, Proc. Natl.Acad. Sci. USA 89, 10915 (1989)).

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gin, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

Specific binding between two entities means an affinity of at least 10⁶,10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰M⁻¹. Affinities greater than 10⁸M⁻¹ arepreferred.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragment including separate heavy chains, lightchains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992).

APP⁶⁹⁵, APP⁷⁵¹, and APP770 refer, respectively, to the 695, 751, and 770amino acid residue long polypeptides encoded by the human APP gene. SeeKang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525(1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids withinthe human amyloid precursor protein (APP) are assigned numbers accordingto the sequence of the APP770 isoform. Terms such as Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43 refer to an Aβ peptide containing amino acid residues1-39, 1-40, 1-41, 1-42 and 1-43.

An “antigen” is an entity to which an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Disaggregated or monomeric Aβ means soluble, monomeric peptide units ofAβ. One method to prepare monomeric Aβ is to dissolve lyophilizedpeptide in neat DMSO with sonication. The resulting solution iscentrifuged to remove any insoluble particulates. Aggregated Aβ is amixture of oligomers in which the monomeric units are held together bynoncovalent bonds.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labelled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibodyis present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises Aβ peptide encompasses both an isolated Aβpeptide and Aβ peptide as a component of a larger polypeptide sequence.

DETAILED DESCRIPTION I. General

Several amyloidogenic diseases and conditions are characterized bypresence of deposits of Aβ peptide aggregated to an insoluble mass inthe brain of a patient. Such diseases include Alzheimer's disease,Down's syndrome and cognitive impairment. The latter is a symptom ofAlzheimer's disease and Down's syndrome but can also without othercharacteristics of either of these diseases. For example, mild cognitiveimpairment or age-associated memory loss occurs in some patient who havenot yet developed, or may never develop full Alzheimer's disease. Mildcognitive impairment can be defined by score on the Mini-Mental StateExam in accordance with convention. Such diseases are characterized byaggregates of Aβ that have a β-pleated sheet structure and stain withCongo Red dye. The basic approach of preventing or treating Alzheimer'sdisease or other amyloidogenic diseases by generating an immunogenicresponse to a component of the amyloid deposit in a patient is describedin WO 99/27944 (incorporated by reference). The present applicationreiterates and confirms the efficacy of the basic approach. The presentapplication is, however, principally directed to improved reagents andmethods. These improvements are premised, in part, on the presentinventors having localized the preferred epitopes within Aβ againstwhich an immunogenic response should be directed. The identification ofpreferred epitopes within Aβ results in agents and methods havingincreased efficacy, reduced potential for side effects, and/or greaterease of manufacture, formulation and administration.

II. Therapeutic Agents

An immunogenic response can be active, as when an immunogen isadministered to induce antibodies reactive with Aβ in a patient, orpassive, as when an antibody is administered that itself binds to Aβ ina patient.

1. Agents Inducing Active Immune Response

Therapeutic agents induce an immunogenic response specifically directedto certain epitopes within Aβ peptides. Preferred agents are the Aβpeptide itself and segments thereof. Variants of such segments, analogsand mimetics of natural Aβ peptide that induce and/or crossreact withantibodies to the preferred epitopes of Aβ peptide can also be used.

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD by interacting with the cleavagereactions by which Aβ is formed so as to increase the amount of the42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to Clq andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

Aβ has several natural occurring forms. The human forms of Aβ arereferred to as Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ42 hasthe sequence:

H₂N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-OH(SEQ ID NO:42).

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-lie,and Ala-fle-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a threonine residue at the C-terminus.

Immunogenic fragments of Aβ are advantageous relative to the intactmolecule in the present methods for several reasons. First, because onlycertain epitopes within Aβ induce a useful immunogenic response fortreatment of Alzheimer's disease, an equal dosage of mass of a fragmentcontaining such epitopes provides a greater molar concentration of theuseful immunogenic epitopes than a dosage of intact Aβ. Second, certainimmunogenic fragments of Aβ generate an immunogenic response againstamyloid deposits without generating a significant immunogenic responseagainst APP protein from which Aβ derives. Third, fragments of Aβ aresimpler to manufacture than intact Aβ due to their shorter size. Fourth,fragments of Aβ do not aggregate in the same manner as intact Aβ,simplifying preparation of pharmaceutical compositions andadministration thereof.

Some immunogenic fragments of Aβ have a sequence of at least 2, 3, 5, 6,10 or 20 contiguous amino acids from a natural peptide. Some immunogenicfragments have no more than 10, 9, 8, 7, 5 or 3 contiguous residues fromAβ. Fragments from the N-terminal half of Aβ are preferred. Preferredimmunogenic fragments include Aβ1-5, 1-6, 1-7, 1-10, 3-7, 1-3, and 1-4.The designation Aβ1-5 for example, indicates a fragment includingresidues 1-5 of Aβ and lacking other residues of Aβ. Fragments beginningat residues 1-3 of Aβ and ending at residues 7-11 of Aβ are particularlypreferred. The fragment Aβ1-12 can also be used but is less preferred.In some methods, the fragment is an N-terminal fragment other thanAβ1-10. Other less preferred fragments include Aβ13-28, 17-28, 1-28,25-35, 35-40 and 35-42. These fragments require screening for activityin clearing or preventing amyloid deposits as described in the Examplesbefore use. Fragments lacking at least one, and sometimes at least 5 or10 C-terminal amino acid present in a naturally occurring forms of Aβare used in some methods. For example, a fragment lacking 5 amino acidsfrom the C-terminal end of Aβ43 includes the first 38 amino acids fromthe N-terminal end of Aβ. Other components of amyloid plaques, forexample, synuclein, and epitopic fragments thereof can also be used toinduce an immunogenic response.

Unless otherwise indicated, reference to Aβ includes the natural humanamino acid sequences indicated above as well as analogs includingallelic, species and induced variants. Analogs typically differ fromnaturally occurring peptides at one, two or a few positions, often byvirtue of conservative substitutions. Analogs typically exhibit at least80 or 90% sequence identity with natural peptides. Some analogs alsoinclude unnatural amino acids or modifications of N or C terminal aminoacids at a one, two or a few positions. For example, the naturalaspartic acid residue at position 1 and/or 7 of Aβ can be replaced withiso-aspartic acid. Examples of unnatural amino acids are D-amino acids,α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid,4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and isoasparticacid. Fragments and analogs can be screened for prophylactic ortherapeutic efficacy in transgenic animal models in comparison withuntreated or placebo controls as described below.

Aβ, its fragments, and analogs can be synthesized by solid phase peptidesynthesis or recombinant expression, or can be obtained from naturalsources. Automatic peptide synthesizers are commercially available fromnumerous suppliers, such as Applied Biosystems, Foster City, Calif.Recombinant expression can be in bacteria, such as E. coli, yeast,insect cells or mammalian cells. Procedures for recombinant expressionare described by Sambrook et al., Molecular Cloning: A Laboratory Manual(C.S.H.P. Press, NY 2d ed., 1989). Some forms of Aβ peptide are alsoavailable commercially (e.g., American Peptides Company, Inc.,Sunnyvale, Claif. and California Peptide Research, Inc. Napa, Calif.).

Therapeutic agents also include longer polypeptides that include, forexample, an active fragment of Aβ peptide, together with other aminoacids. For example, preferred agents include fusion proteins comprisinga segment of Aβ fused to a heterologous amino acid sequence that inducesa helper T-cell response against the heterologous amino acid sequenceand thereby a B-cell response against the Aβ segment. Such polypeptidescan be screened for prophylactic or therapeutic efficacy in animalmodels in comparison with untreated or placebo controls as describedbelow. The Aβ peptide, analog, active fragment or other polypeptide canbe administered in associated or multimeric form or in dissociated formTherapeutic agents also include multimers of monomeric immunogenicagents.

In a further variation, an immunogenic peptide, such as a fragment ofAβ, can be presented by a virus or a bacteria as part of an immunogeniccomposition. A nucleic acid encoding the immunogenic peptide isincorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of a bacteria so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable. Therapeutic agents also include peptidesand other compounds that do not necessarily have a significant aminoacid sequence similarity with Aβ but nevertheless serve as mimetics ofAβ and induce a similar immune response. For example, any peptides andproteins forming β-pleated sheets can be screened for suitability.Anti-idiotypic antibodies against monoclonal antibodies to Aβ or otheramyloidogenic peptides can also be used. Such anti-Id antibodies mimicthe antigen and generate an immune response to it (see EssentialImmunology (Roit ed., Blackwell Scientific Publications, Palo Alto, 6thed.), p. 181). Agents other than Aβ peptides should induce animmunogenic response against one or more of the preferred segments of Aβlisted above (e.g., 1-10, 1-7, 1-3, and 3-7). Preferably, such agentsinduce an immunogenic response that is specifically directed to one ofthese segments without being directed to other segments of Aβ.

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for Aβ or other amyloidogenicpeptides. For example, initial screens can be performed with anypolyclonal sera or monoclonal antibody to Aβ or a fragment thereof.Compounds can then be screened for binding to a specific epitope withinAβ (e.g., 1-10, 1-7, 1-3, 1-4, 1-5 and 3-7). Compounds can be tested bythe same procedures described for mapping antibody epitopespecificities. Compounds identified by such screens are then furtheranalyzed for capacity to induce antibodies or reactive lymphocytes to Aβor fragments thereof. For example, multiple dilutions of sera can betested on microtiter plates that have been precoated with Aβ or afragment thereof and a standard ELISA can be performed to test forreactive antibodies to Aβ or the fragment. Compounds can then be testedfor prophylactic and therapeutic efficacy in transgenic animalspredisposed to an amyloidogenic disease, as described in the Examples.Such animals include, for example, mice bearing a 717 mutation of APPdescribed by Games et al., supra, and mice bearing a 670/671 Swedishmutation of APP such as described by McConlogue et al., U.S. Pat. No.5,612,486 and Hsiao et al., Science 274, 99 (1996); Staufenbiel et al.,Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Sturchler-Pierrat etal., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Borchelt et al.,Neuron 19, 939-945 (1997)). The same screening approach can be used onother potential agents analogs of Aβ and longer peptides includingfragments of Aβ, described above.

2. Agents Inducing Passive Immune Response

Therapeutic agents of the invention also include antibodies thatspecifically bind to Aβ or other component of amyloid plaques. Suchantibodies can be monoclonal or polyclonal. Some such antibodies bindspecifically to the aggregated form of Aβ without binding to thedissociated form. Some bind specifically to the dissociated form withoutbinding to the aggregated form. Some bind to both aggregated anddissociated forms. Some such antibodies bind to a naturally occurringshort form of Aβ (i.e., Aβ39, 40 or 41) without binding to a naturallyoccurring long form of Aβ (i.e., Aβ42 and Aβ43). Some antibodies bind toa long form without binding to a short form. Some antibodies bind to Aβwithout binding to full-length amyloid precursor protein. Antibodiesused in therapeutic methods usually have an intact constant region or atleast sufficient of the constant region to interact with an Fc receptor.Human isotype IgG1 is preferred because of it having highest affinity ofhuman isotypes for the FcRI receptor on phagocytic cells. Bispecific Fabfragments can also be used, in which one arm of the antibody hasspecificity for Aβ, and the other for an Fc receptor. Some antibodiesbind to Aβ with a binding affinity greater than or equal to about 10⁶,10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of Aβ. However, polyclonalsera can be specific to a particular segment of Aβ, such as Aβ1-10.Monoclonal antibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples. Preferred monoclonalantibodies bind to an epitope within residues 1-10 of Aβ (with the firstN terminal residue of natural Aβ designated 1). Some preferredmonoclonal antibodies bind to an epitope within amino acids 1-5, andsome to an epitope within 5-10. Some preferred antibodies bind toepitopes within amino acids 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7. Somepreferred antibodies bind to an epitope starting at resides 1-3 andending at residues 7-11 of Aβ. Less preferred antibodies include thosebinding to epitopes with residues 10-15, 15-20, 25-30, 10-20, 20, 30, or10-25 of Aβ. It is recommended that such antibodies be screened foractivity in the mouse model described in the Examples before use. Forexample, it has been found that certain antibodies to epitopes withinresidues 10-18, 16-24, 18-21 and 33-42 lack activity. In some methods,multiple monoclonal antibodies having binding specificities to differentepitopes are used. Such antibodies can be administered sequentially orsimultaneously. Antibodies to amyloid components other than Aβ can alsobe used. For example, antibodies can be directed to the amyloidassociated protein synuclein.

When an antibody is said to bind to an epitope within specifiedresidues, such as Aβ1-5 for example, what is meant is that the antibodyspecifically binds to a polypeptide containing the specified residues(i.e., Aβ1-5 in this an example). Such an antibody does not necessarilycontact every residue within Aβ1-5. Nor does every single amino acidsubstitution or deletion with in Aβ1-5 necessarily significantly affectbinding affinity. Epitope specificity of an antibody can be determined,for example, by forming a phage display library in which differentmembers display different subsequences of Aβ. The phage display libraryis then selected for members specifically binding to an antibody undertest. A family of sequences is isolated. Typically, such a familycontains a common core sequence, and varying lengths of flankingsequences in different members. The shortest core sequence showingspecific binding to the antibody defines the epitope bound by theantibody. Antibodies can also be tested for epitope specificity in acompetition assay with an antibody whose epitope specificity has alreadybeen determined. For example, antibodies that compete with the 3D6antibody for binding to Aβ bind to the same or similar epitope as 3D6,i.e., within residues Aβ1-5. Likewise antibodies that compete with the10D5 antibody bind to the same or similar epitope, i.e., within residuesAβ3-6. Screening antibodies for epitope specificity is a usefulpredictor of therapeutic efficacy. For example, an antibody determinedto bind to an epitope within residues 1-7 of Aβ is likely to beeffective in preventing and treating Alzheimer's disease.

Monoclonal or polyclonal antibodies that specifically bind to apreferred segment of Aβ without binding to other regions of Aβ have anumber of advantages relative to monoclonal antibodies binding to otherregions or polyclonal sera to intact Aβ. First, for equal mass dosages,dosages of antibodies that specifically bind to preferred segmentscontain a higher molar dosage of antibodies effective in clearingamyloid plaques. Second, antibodies specifically binding to preferredsegments can induce a clearing response against amyloid deposits withoutinducing a clearing response against intact APP polypeptide, therebyreducing the potential for side effects.

i. General Characteristics of Immunoglobulins

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917(1987);Chothia et al., Nature 342:878-883 (1989).

ii. Production of Nonhuman Antibodies

The production of non-human monoclonal antibodies, e.g., murine, guineapig, primate, rabbit or rat, can be accomplished by, for example,immunizing the animal with Aβ. A longer polypeptide comprising Aβ or animmunogenic fragment of Aβ or anti-idiotypic antibodies to an antibodyto Aβ can also be used. See Harlow & Lane, Antibodies, A LaboratoryManual (CSHP NY, 1988) (incorporated by reference for all purposes).Such an immunogen can be obtained from a natural source, by peptidesynthesis or by recombinant expression. Optionally, the immunogen can beadministered fused or otherwise complexed with a carrier protein, asdescribed below. Optionally, the immunogen can be administered with anadjuvant. Several types of adjuvant can be used as described below.Complete Freund's adjuvant followed by incomplete adjuvant is preferredfor immunization of laboratory animals. Rabbits or guinea pigs aretypically used for making polyclonal antibodies. Mice are typically usedfor making monoclonal antibodies. Antibodies are screened for specificbinding to Aβ. Optionally, antibodies are further screened for bindingto a specific region of Aβ. The latter screening can be accomplished bydetermining binding of an antibody to a collection of deletion mutantsof an Aβ peptide and determining which deletion mutants bind to theantibody. Binding can be assessed, for example, by Western blot orELISA. The smallest fragment to show specific binding to the antibodydefines the epitope of the antibody. Alternatively, epitope specificitycan be determined by a competition assay is which a test and referenceantibody compete for binding to Aβ. If the test and reference antibodiescompete, then they bind to the same epitope or epitopes sufficientlyproximal that binding of one antibody interferes with binding of theother. The preferred isotype for such antibodies is mouse isotype IgG2aor equivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1.

iii. Chimeric and Humanized Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. Human isotype IgG1 is preferred. A typicalchimeric antibody is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse antibody and the C or effectordomain from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc. NatlAcad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. Nos.5,693,762, 5,693,761, 5,585,089, 5,530,101 and Winter, U.S. Pat. No.5,225,539 (incorporated by reference in their entirety for allpurposes). The constant region(s), if present, are also substantially orentirely from a human immunoglobulin. The human variable domains areusually chosen from human antibodies whose framework sequences exhibit ahigh degree of sequence identity with the murine variable region domainsfrom which the CDRs were derived. The heavy and light chain variableregion framework residues can be derived from the same or differenthuman antibody sequences. The human antibody sequences can be thesequences of naturally occurring human antibodies or can be consensussequences of several human antibodies. See Carter et al., WO 92/22653.Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. Investigation of such possibleinfluences is by modeling, examination of the characteristics of theamino acids at particular locations, or empirical observation of theeffects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g. is within about 6 A of aCDR region), or

(4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

iv. Human Antibodies

Human antibodies against Aβ are provided by a variety of techniquesdescribed below. Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody, such as one of the mouse monoclonalsdescribed in Example XI. Human antibodies can also be screened for aparticular epitope specificity by using only a fragment of Aβ as theimmunogen, and/or by screening antibodies against a collection ofdeletion mutants of Aβ. Human antibodies preferably have isotypespecificity human IgG1.

(1) Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman etal., U.S. Pat. No. 4,634,666 (each of which is incorporated by referencein its entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells—two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with Aβ, a fragment thereof, larger polypeptidecontaining Aβ or fragment, or an anti-idiotypic antibody to an antibodyto Aβ. In some methods, B cells are isolated from the same patient whois ultimately to be administered antibody therapy. For in vitroimmunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C, forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof. Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

(2) Transgenic Non-Human Mammals

Human antibodies against Aβ can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., W093/12227 (1993);U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429,5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148,1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO91/10741 (1991) (each of which is incorporated by reference in itsentirety for all purposes). Transgenic mice are particularly suitable.Anti-Aβ antibodies are obtained by immunizing a transgenic nonhumanmammal, such as described by Lonberg or Kucherlapati, supra, with Aβ ora fragment thereof. Monoclonal antibodies are prepared by, e.g., fusingB-cells from such mammals to suitable myeloma cell lines usingconventional Kohler-Milstein technology. Human polyclonal antibodies canalso be provided in the form of serum from humans immunized with animmunogenic agent. Optionally, such polyclonal antibodies can beconcentrated by affinity purification using Aβ or other amyloid peptideas an affinity reagent.

(3) Phage Display Methods

A further approach for obtaining human anti-Aβ antibodies is to screen aDNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). As described fortrioma methodology, such B cells can be obtained from a human immunizedwith Aβ, fragments, longer polypeptides containing Aβ or fragments oranti-idiotypic antibodies. Optionally, such B cells are obtained from apatient who is ultimately to receive antibody treatment. Antibodiesbinding to Aβ or a fragment thereof are selected. Sequences encodingsuch antibodies (or a binding fragments) are then cloned and amplified.The protocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. Nos. 5,877,218, 5,871,907,5,858,657, 5,837,242, 5,733,743 and 5,565,332 (each of which isincorporated by reference in its entirety for all purposes). In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to an Aβ peptideor fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for Aβ(e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) is selected. Thehuman heavy chain variable region from this phage then serves as astarting material for constructing a further phage library. In thislibrary, each phage displays the same heavy chain variable region (i.e.,the region identified from the first display library) and a differentlight chain variable region. The light chain variable regions areobtained from a library of rearranged human variable light chainregions. Again, phage showing strong specific binding for Aβ areselected. These phage display the variable regions of completely humananti-Aβ antibodies. These antibodies usually have the same or similarepitope specificity as the murine starting material.

v. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Human isotype IgG1 is preferred.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

vi. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells and myeloma cell lines. Preferably, the cellsare nonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957, 5,304,489, 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains in operablelinkage with a promoter and enhancer from a mammary gland specific gene,such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

3. Carrier Proteins

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against amyloid deposits but aretoo small to be immunogenic. In this situation, a peptide immunogen canbe linked to a suitable carrier to help elicit an immune response.Suitable carriers include serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or atoxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. Other carriersinclude T-cell epitopes that bind to multiple MHC alleles, e.g., atleast 75% of all human MHC alleles. Such carriers are sometimes known inthe art as “universal T-cell epitopes.” Examples of universal T-cellepitopes include:

Influenza Hemagluttinin: HA₃₀₇₋₃₁₉ PKYVKQNTLKLAT (SEQ ID NO:43)

PADRE (common residues bolded) AKXVAAWTLKAAA (SEQ ID NO:44)

Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO:45)

Hepatitis B surface antigen: HBsAg₁₉₋₂₈ FFLLTRILTI (SEQ ID NO:46)

Heat Shock Protein 65: hsp65₁₅₃₋₁₇₁ DQSIGDLIAEAMDKVGNEG (SEQ ID NO:47)

bacille Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO:48)

Tetanus toxoid: TT₈₃₀₋₈₄₄ QYIKANSKFIGITEL (SEQ ID NO:49)

Tetanus toxoid: TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:50)

HIV gp120 T1: KQIINMWQEVGKAMYA (SEQ ID NO:51).

Other carriers for stimulating or enhancing an immune response includecytokines such as IL-1, IL-1 α and β peptides, IL-2, γINF, IL-10,GM-CSF, and chemokines, such as MIP1α and β and RANTES. Immunogenicagents can also be linked to peptides that enhance transport acrosstissues, as described in O'Mahony, WO 97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the ε-amino on a lysine, or other freeamino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fuision protein. Optionally, an immunogenic peptide canbe linked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Some carrier peptides serve toinduce a helper T-cell response against the carrier peptide. The inducedhelper T-cells in turn induce a B-cell response against the immunogenicpeptide linked to the carrier peptide.

Some agents of the invention comprise a fusion protein in which anN-terminal fragment of Aβ is linked at its C-terminus to a carrierpeptide. In such agents, the N-terminal residue of the fragment of Aβconstitutes the N-terminal residue of the fusion protein. Accordingly,such fusion proteins are effective in inducing antibodies that bind toan epitope that requires the N-terminal residue of Aβ to be in freeform. Some agents of the invention comprises a plurality of repeats ofan N-terminal segment of Aβ linked at the C-terminus to one or more copyof a carrier peptide. The N-terminal fragment of Aβ incorporated intosuch fusion proteins sometimes begins at Aβ1-3 and ends at Aβ7-1 1.Aβ1-7, Aβ1-3, 1-4, 1-5, and 3-7 are preferred N-terminal fragment Aβ.Some fusion proteins comprise different N-terminal segments of Aβ intandem. For example, a fusion protein can comprise Aβ1-7 followed byAβ1-3 followed by a heterologous peptide.

In some fusion proteins, an N-terminal segment of Aβ is fused at itsN-terminal end to a heterologous carrier peptide. The same variety ofN-terminal segments of Aβ can be used as with C-terminal fusions. Somefusion proteins comprise a heterologous peptide linked to the N-terminusof an N-terminal segment of Aβ, which is in turn linked to one or moreadditional N-terminal segments of Aβ in tandem.

Some examples of fusion proteins suitable for use in the invention areshown below. Some of these fusion proteins comprise segments of Aβlinked to tetanus toxoid epitopes such as described in U.S. Pat. No.5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprisessegments of Aβ linked to carrier peptides described in U.S. Pat. No.5,736,142. Some heterologous peptides are universal T-cell epitopes. Insome methods, the agent for administration is simply a single fusionprotein with an Aβ segment linked to a heterologous segment in linearconfiguration. In some methods, the agent is multimer of fusion proteinsrepresented by the formula 2^(x), in which x is an integer from 1-5.Preferably x is 1, 2 or 3, with 2 being most preferred. When x is two,such a multimer has four fusion proteins linked in a preferredconfiguration referred to as MAP4 (see U.S. Pat. No. 5,229,490).Epitopes of Aβ are underlined.

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N termini. In this example, fouridentical N termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells.

AN90549 (Aβ1-7/Tetanus toxoid 830-844 in a MAP4 configuration):

DAEFRHDQYIKANSKFIGITEL (SEQ ID NO:52)

AN90550 (Aβ1-7/Tetanus toxoid 947-967 in a MAP4 configuration):

DAEFRHDFNNFTVSFWLRVPKVSASHLE (SEQ ID NO:53)

AN90542 (Aβ1-7/Tetanus toxoid 830-844+947-967 in a linearconfiguration):

DAEFRHDQYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE (SEQ ID NO:54)

AN90576: (Aβ3-9)/Tetanus toxoid 830-844 in a MAP4 configuration):

EFRHDSGQYIKANSKFIGITEL (SEQ ID NO:55)

Peptide described in U.S. Pat. No. 5,736,142 (all in linearconfigurations):

AN90562 (Aβ1-7/peptide) AKXVAAWTLKAAADAEFRHD (SEQ ID NO:56)

AN90543 (Aβ1-7×3/ peptide): DAEFRHDDAEFRHDDAEFRHDAKXVAAWTLKAAA (SEQ IDNO:57)

Other examples of fuision proteins (immunogenic epitope of Aβ bolded)include

AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD DAEFRHD-AKXVAAWTLKAAA (SEQ IDNO:59)

DAEFRHD-ISQAVHAAHAEINEAGR (SEQ ID NO:60)

FRHDSGY-ISQAVHAAHAEINEAGR (SEQ ID NO:61)

EFRHDSG-ISQAVHAAHAEINEAGR (SEQ ID NO:62)

PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO:63)

DAEFRHD-PKYVKQNTLKLAT-DAEFRHD (SEQ ID NO:64)

DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO:65)

DAEFRHD-DAEFRHD-PKYVKQNTLKLAT (SEQ ID NO:66)

DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-DAEFRHD(SEQ ID NO:67)

DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE (SEQ IDNO:68)

DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE (SEQ ID NO:69)

DAEFRHD-QY KANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-DAEFRHD (SEQ ID NO:70)

DAEFRHD-QYIKANSKFIGITEL on a 2 branched resin (SEQ ID NO:77)

EQVTNVGGAISQAVHAAHAEINEAGR (SEQ ID NO:71) (Synuclein fusion protein inMAP4 configuration).

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstAβ for use in passive immunization. For example, Aβ or a fragment linkedto a carrier can be administered to a laboratory animal in theproduction of monoclonal antibodies to Aβ.

4. Nucleic Acid Encoding Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding segments of Aβ peptide, andfragments thereof, other peptide immunogens, or antibodies and theircomponent chains used for passive immunization. Such nucleic acids canbe DNA or RNA. A nucleic acid segment encoding an immunogen is typicallylinked to regulatory elements, such as a promoter and enhancer, thatallow expression of the DNA segment in the intended target cells of apatient. For expression in blood cells, as is desirable for induction ofan immune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO96/34625)and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333(1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res.24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). Such vectorscan further include facilitating agents such as bupivacine (U.S. Pat.No. 5,593,970). DNA can also be administered using a gene gun. See Xiao& Brandsma, supra. The DNA encoding an immunogen is precipitated ontothe surface of microscopic metal beads. The microprojectiles areaccelerated with a shock wave or expanding helium gas, and penetratetissues to a depth of several cell layers. For example, The Accel™ GeneDelivery Device manufactured by Agacetus, Inc. Middleton Wis. issuitable. Alternatively, naked DNA can pass through skin into the bloodstream simply by spotting the DNA onto skin with chemical or mechanicalirritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

III. Screening Antibodies for Clearing Activity

The invention provides methods of screening an antibody for activity inclearing an amyloid deposit or any other antigen, or associatedbiological entity, for which clearing activity is desired. To screen foractivity against an amyloid deposit, a tissue sample from a brain of apatient with Alzheimer's disease or an animal model havingcharacteristic Alzbeimer's pathology is contacted with phagocytic cellsbearing an Fc receptor, such as microglial cells, and the antibody undertest in a medium in vitro. The pagocytic cells can be a primary cultureor a cell line, such as BV-2, C8-B4, or THP-1. In some methods, thecomponents are combined on a microscope slide to facilitate microscopicmonitoring. In some methods, multiple reactions are performed inparallel in the wells of a microtiter dish. In such a format, a separateminiature microscope slide can be mounted in the separate wells, or anonmicroscopic detection format, such as ELISA detection of Aβ can beused. Preferably, a series of measurements is made of the amount ofamyloid deposit in the in vitro reaction mixture, starting from abaseline value before the reaction has proceeded, and one or more testvalues during the reaction. The antigen can be detected by staining, forexample, with a fluorescently labelled antibody to Aβ or other componentof amyloid plaques. The antibody used for staining may or may not be thesame as the antibody being tested for clearing activity. A reductionrelative to baseline during the reaction of the amyloid depositsindicates that the antibody under test has clearing activity. Suchantibodies are likely to be useful in preventing or treating Alzheimer'sand other amyloidogenic diseases.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used include Aβ,viral antigens or viruses, proteoglycans, antigens of other pathogenicmicroorganisms, tumor antigens, and adhesion molecules. Such antigenscan be obtained from natural sources, recombinant expression or chemicalsynthesis, among other means. The tissue sample or isolated biologicalentity is contacted with phagocytic cells bearing Fc receptors, such asmonocytes or microglial cells, and an antibody to be tested in a medium.The antibody can be directed to the biological entity under test or toan antigen associated with the entity In the latter situation, theobject is to test whether the biological entity is vicariouslyphagocytosed with the antigen. Usually, although not necessarily, theantibody and biological entity (sometimes with an associated antigen)are contacted with each other before adding the phagocytic cells. Theconcentration of the biological entity and/or the associated antigen, ifpresent, remaining in the medium is then monitored. A reduction in theamount or concentration of antigen or the associated biological entityin the medium indicates the antibody has a clearing response against theantigen and/or associated biological entity in conjunction with thephagocytic cells (see, e.g., Example 14).

IV. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who do have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy,TINS, supra). Other markers of risk are mutations in the presenilingenes, PS1 and PS2, and ApoE4, family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized from characteristic dementia,as well as the presence of risk factors described above. In addition, anumber of diagnostic tests are available for identifying individuals whohave AD. These include measurement of CSF tau and Aβ42 levels. Elevatedtau and decreased Aβ42 levels signify the presence of AD. Individualssuffering from Alzheimer's disease can also be diagnosed by ADRDAcriteria as discussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., Aβ peptide) over time. If the response falls, a boosterdosage is indicated. In the case of potential Down's syndrome patients,treatment can begin antenatally by administering therapeutic agent tothe mother or shortly after birth.

V. Treatment Regimes

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, compositions ormedicants are administered to a patient suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Insome methods, administration of agent reduces or eliminates myocognitiveimpairment in patients that have not yet developed characteristicAlzheimer's pathology. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1-500 μg per patient and more usually from 5-500 μg per injectionfor human administration. Occasionally, a higher dose of 1-2 mg perinjection is used. Typically about 10, 20, 50 or 100 μg is used for eachhuman injection. The mass of immunogen also depends on the mass ratio ofimmunogenic epitope within the immunogen to the mass of immunogen as awhole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitope areused for microgram of immunogen. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/ patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals. Another regimen consists of animmunization followed by booster injections 1, 2 and 12 months later.Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per every two weeks or once a month or onceevery 3 to 6 months. In some methods, two or more monoclonal antibodieswith different binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of antibody to Aβ in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 ug/ml andin some methods 25-300 ug/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal or intramuscular means forprophylactic and/or therapeutic treatment. The most typical route ofadministration of an immunogenic agent is subcutaneous although otherroutes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. Intramuscular injectionon intravenous infusion are preferred for administration of antibody. Insome methods, particular therapeutic antibodies are injected directlyinto the cranium. In some methods, antibodies are administered as asustained release composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as Aβ, to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealuminum hydroxide and aluminum phosphate, 3 De-O-acylatedmonophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Mont., now part of Corixa). Stimulon™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540),(AquilaBioPhannaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (WO98/40100). Alternatively, Aβ can be coupled to an adjuvant. However,such coupling should not substantially change the conformation of Aβ soas to affect the nature of the immune response thereto. Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS-21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine: Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another classof preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21,Aquila, Framingham, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Other adjuvants include cytokines, such as interleukins(IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF).

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. Alum, MPL and QS-21 are preferred.Optionally, two or more different adjuvants can be used simultaneously.Preferred combinations include alum with MPL, alum with QS-21, MPL withQS-21, and alum, QS-21 and MPL together. Also, Incomplete Freund'sadjuvant can be used (Chang et al., Advanced Drug Delivery Reviews 32,173-186 (1998)), optionally in combination with any of alum, QS-21, andMPL and all combinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized sepharose(™), agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes). Additionally, these carriers can functionas immunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles priorto injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

VI. Methods of Diagnosis

The invention provides methods of detecting an immune response againstAβ peptide in a patient suffering from or susceptible to Alzheimer'sdisease. The methods are particularly useful for monitoring a course oftreatment being administered to a patient. The methods can be used tomonitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. The methods are usefulfor monitoring both active immunization (e.g., antibody produced inresponse to administration of immunogen) and passive immunization (e.g.,measuring level of administered antibody).

1. Active Immunization

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an immunogenic agent are expected toshow an increase in immune response with successive dosages, whicheventually reaches a plateau. Administration of agent is generallycontinued while the immune response is increasing. Attainment of theplateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have plateaued in response to treatment. Measuredvalues of immune response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different(e.g., more than one standard deviation) from the control value,treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to any form of Aβ peptide, typicallyAβ42. The immune response can be determined from the presence of, e.g.,antibodies or T-cells that specifically bind to Aβ peptide. ELISAmethods of detecting antibodies specific to Aβ are described in theExamples section. Methods of detecting reactive T-cells have beendescribed above (see Definitions). In some methods, the immune responseis determined using a clearing assay, such as described in Section IIIabove. In such methods, a tissue sample from a patient being tested iscontacted with amyloid deposits (e.g., from a PDAPP mouse) andphagocytic cells bearing Fc receptors. Subsequent clearing of theamyloid deposit is then monitored. The existence and extent of clearingresponse provides an indication of the existence and level of antibodieseffective to clear Aβ in the tissue sample of the patient under test.

2. Passive Immunization

In general, the procedures for monitoring passive immunization aresimilar to those for monitoring active immunization described above.However, the antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

3. Diagnostic Kits

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to Aβ. The kit can alsoinclude a label. For detection of antibodies to Aβ, the label istypically in the form of labelled anti-idiotypic antibodies. Fordetection of antibodies, the agent can be supplied prebound to a solidphase, such as to the wells of a microtiter dish. Kits also typicallycontain labeling providing directions for use of the kit. The labelingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to Aβ. The termlabeling refers to any written or recorded material that is attached to,or otherwise accompanies a kit at any time during its manufacture,transport, sale or use. For example, the term labeling encompassesadvertising leaflets and brochures, packaging materials, instructions,audio or video cassettes, computer discs, as well as writing imprinteddirectly on kits.

The invention also provides diagnostic kits for performing in vivoimaging. Such kits typically contain an antibody binding to an epitopeof Aβ, preferably within residues 1-10. Preferably, the antibody islabelled or a secondary labeling reagent is included in the kit.Preferably, the kit is labelled with instructions for performing an invivo imaging assay.

VII. In Vivo Imaging

The invention provides methods of in vivo imaging amyloid deposits in apatient. Such methods are useful to diagnose or confirm diagnosis ofAlzheimer's disease, or susceptibility thereto. For example, the methodscan be used on a patient presenting with symptoms of dementia. If thepatient has abnormal amyloid deposits, then the patient is likelysuffering from Alzheimer's disease. The methods can also be used onasymptomatic patients. Presence of abnormal deposits of amyloidindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed withAlzheimer's disease.

The methods work by administering a reagent, such as antibody, thatbinds to Aβ in the patient, and then detecting the agent after it hasbound. Preferred antibodies bind to Aβ deposits in a patient withoutbinding to full length APP polypeptide. Antibodies binding to an epitopeof Aβ within amino acids 1-10 are particularly preferred. In somemethods, the antibody binds to an epitope within amino acids 7-10 of Aβ.Such antibodies typically bind without inducing a substantial clearingresponse. In other methods, the antibody binds to an epitope withinamino acids 1-7 of Aβ. Such antibodies typically bind and induce aclearing response to Aβ. However, the clearing response can be avoidedby using antibody fragments lacking a full length constant region, suchas Fabs. In some methods, the same antibody can serve as both atreatment and diagnostic reagent. In general, antibodies binding toepitopes C-terminal of residue 10 of Aβ do not show as strong signal asantibodies binding to epitopes within residues 1-10, presumably becausethe C-terminal epitopes are inaccessible in amyloid deposits.Accordingly, such antibodies are less preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labelled, although in some methods, the primary reagentwith affinity for Aβ is unlabelled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabelled loci to corresponding base line values. The base line valuescan represent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

EXAMPLES 1. Prophylactic Efficacy of Aβ Against AD

These examples describe administration of Aβ42 peptide to transgenicmice overexpressing APP with a mutation at position 717 (APP_(717v→F))that predisposes them to develop Alzheimer's-like neuropathology.Production and characteristics of these mice (PDAPP mice) is describedin Games et al., Nature, supra. These animals, in their heterozygoteform, begin to deposit Aβ at six months of age forward. By fifteenmonths of age they exhibit levels of Aβ deposition equivalent to thatseen in Alzheimer's disease. PDAPP mice were injected with aggregatedAβ₄₂ (aggregated Aβ₄₂) or phosphate buffered saline. Aggregated Aβ₄₂ waschosen because of its ability to induce antibodies to multiple epitopesof Aβ.

A. Methods

1. Source of Mice

Thirty PDAPP heterogenic female mice were randomly divided into thefollowing groups: 10 mice to be injected with aggregated Aβ42 (one diedin transit), 5 mice to be injected with PBS/adjuvant or PBS, and 10uninjected controls. Five mice were injected with peptides derived fromthe sequence of serum amyloid protein (SAP).

2. Preparation of Immunogens

Preparation of aggregated Aβ42: two milligrams of Aβ42 (U.S. PeptidesInc, lot K-42-12) was dissolved in 0.9 ml water and made up to 1 ml byadding 0.1 ml 10 ×PBS. This was vortexed and allowed to incubateovernight 37° C., under which conditions the peptide aggregated. Anyunused Aβ was stored as a dry lyophilized powder at −20° C. until thenext injection.

3. Preparation of Injections

For each injection, 100 μg of aggregated Aβ42 in PBS per mouse wasemulsified 1:1 with Complete Freund's adjuvant (CFA) in a final volumeof 400 μl emulsion for the first immunization, followed by a boost ofthe same amount of immunogen in Incomplete Freund's adjuvant (IFA) at 2weeks. Two additional doses in IFA were given at monthly intervals. Thesubsequent immunizations were done at monthly intervals in 500 μl ofPBS. Injections were delivered intraperitoneally (i.p.).

PBS injections followed the same schedule and mice were injected with a1:1 mix of PBS/ Adjuvant at 400 μl per mouse, or 500 μl of PBS permouse. SAP injections likewise followed the same schedule using a doseof 100 μg per injection.

4. Titration of Mouse Bleeds, Tissue Preparation andImmunohistochemistry

The above methods are described infra in General Materials and Methods.

B. Results

PDAPP mice were injected with either aggregated Aβ42 (aggregated Aβ42),SAP peptides, or phosphate buffered saline. A group of PDAPP mice werealso left as uninjected, positive controls. The titers of the mice toaggregated Aβ42 were monitored every other month from the fourth boostuntil the mice were one year of age. Mice were sacrificed at 13 months.At all time points examined, eight of the nine aggregated Aβ42 micedeveloped a high antibody titer, which remained high throughout theseries of injections (titers greater than 1/10000). The ninth mouse hada low, but measurable titer of approximately 1/1000 (FIG. 1, Table 1).SAPP-injected mice had titers of 1:1,000 to 1:30,000 for this immunogenwith only a single mouse exceeding 1:10,0000.

The PBS-treated mice were titered against aggregated Aβ42 at six, tenand twelve months. At a 1/100 dilution the PBS mice, when titeredagainst aggregated Aβ42, only exceeded 4 times background at one datapoint, otherwise, they were less than 4 times background at all timepoints (Table 1). The SAP-specific response was negligible at these timepoints with all titers less than 300.

Seven out of the nine mice in the aggregated Aβ1-42 treated group had nodetectable amyloid in their brains. In contrast, brain tissue from micein the SAP and PBS groups contained numerous amyloid deposits in thehippocampus, as well as in the frontal and cingulate cortices. Thepattern of deposition was similar to that of untreated controls, withcharacteristic involvement of vulnerable subregions, such as the outermolecular layer of the hippocampal dentate gyrus. One mouse from theAβ1-42-injected group had a greatly reduced amyloid burden, confined tothe hippocampus. An isolated plaque was identified in anotherAβ1-42-treated mouse.

Quantitative image analyses of the amyloid burden in the hippocampusverified the dramatic reduction achieved in the Aβ42(AN1792)-treatedanimals (FIG. 2). The median values of the amyloid burden for the PBSgroup (2.22%), and for the untreated control group (2.65%) weresignificantly greater than for those immunized with AN1792 (0.00%,p=0.0005). In contrast, the median value for the group immunized withSAP peptides (SAPP) was 5.74%. Brain tissue from the untreated, controlmice contained numerous Aβ amyloid deposits visualized with the AP-specific monoclonal antibody (mAb) 3D6 in the hippocampus, as well asin the retrosplenial cortex. A similar pattern of amyloid deposition wasalso seen in mice immunized with SAPP or PBS (FIG. 2). In addition, inthese latter three groups there was a characteristic involvement ofvulnerable subregions of the brain classically seen in Aβ, such as theouter molecular layer of the hippocampal dentate gyrus, in all three ofthese groups.

The brains that contained no Aβ deposits were also devoid of neuriticplaques that are typically visualized in PDAPP mice with the human APPantibody 8E5. All of brains from the remaining groups (SAP-injected, PBSand uninjected mice) had numerous neuritic plaques typical of untreatedPDAPP mice. A small number of neuritic plaques were present in one mousetreated with AN1792, and a single cluster of dystrophic neurites wasfound in a second mouse treated with AN1792. Image analyses of thehippocampus, and shown in FIG. 3, demonstrated the virtual eliminationof dystrophic neurites in AN1792-treated mice (median 0.00%) compared tothe PBS recipients (median 0.28%, p=0.0005).

Astrocytosis characteristic of plaque-associated inflammation was alsoabsent in the brains of the Aβ1-42 injected group. The brains from themice in the other groups contained abundant and clustered GFAP-positiveastrocytes typical of Aβ plaque-associated gliosis. A subset of theGFAP-reacted slides were counter-stained with Thioflavin S to localizethe Aβ deposits. The GFAP-positive astrocytes were associated with Aβplaques in the SAP, PBS and untreated controls. No such association wasfound in the plaque-negative Aβ1-42 treated mice, while minimalplaque-associated gliosis was identified in one mouse treated withAN1792.

Image analyses, shown in FIG. 4 for the retrosplenial cortex, verifiedthat the reduction in astrocytosis was significant with a median valueof 1.56% for those treated with AN1792 versus median values greater than6% for groups immunized with SAP peptides, PBS or untreated (p=0.0017).

Evidence from a subset of the Aβ1-42- and PBS-injected mice indicatedplaque-associated MHC II immunoreactivity was absent in the Aβ1-42injected mice, consistent with lack of an Aβ-related inflammatoryresponse.

Sections of the mouse brains were also reacted with a mAb specific witha monoclonal antibody specific for MAC-1, a cell surface protein. MAC-1(CD11b) is an integrin family member and exists as a heterodimer withCD18. The CD11b/CD18 complex is present on monocytes, macrophages,neutrophils and natural killer cells (Mak and Simard). The residentMAC-1-reactive cell type in the brain is likely to be microglia based onsimilar phenotypic morphology in MAC-1 immunoreacted sections.Plaque-associated MAC-1 labeling was lower in the brains of mice treatedwith AN1792 compared to the PBS control group, a finding consistent withthe lack of an Aβ-induced inflammatory response.

C. Conclusion

The lack of Aβ plaques and reactive neuronal and gliotic changes in thebrains of the Aβ1-42-injected mice indicate that no or extremely littleamyloid was deposited in their brains, and pathological consequences,such as gliosis and neuritic pathology, were absent. PDAPP mice treatedwith Aβ1-42 show essentially the same lack of pathology as controlnontransgenic mice. Therefore, Aβ1-42 injections are highly effective inthe prevention of deposition or clearance of human Aβ from brain tissue,and elimination of subsequent neuronal and inflammatory degenerativechanges. Thus, administration of Aβ peptide can have both preventativeand therapeutic benefit in prevention of Aβ.

II. Dose Response Study

Groups of five-week old, female Swiss Webster mice (N=6 per group) wereimmunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13 ug of Aβformulated in CFA/IFA administered intraperitoneally. Three doses weregiven at biweekly intervals followed by a fourth dose one month later.The first dose was emulsified with CFA and the remaining doses wereemulsified with IFA. Animals were bled 4-7 days following eachimmunization starting after the second dose for measurement of antibodytiters. Animals in a subset of three groups, those immunized with 11,33, or 300 ug of antigen, were additionally bled at approximatelymonthly intervals for four months following the fourth immunization tomonitor the decay of the antibody response across a range of doses ofimmunogenic formulations. These animals received a final fifthimmunization at seven months after study initiation. They weresacrificed one week later to measure antibody responses to AN1792 and toperform toxicological analyses.

A declining dose response was observed from 300 to 3.7 μg with noresponse at the two lowest doses. Mean antibody titers are about 1:1000after 3 doses and about 1:10,000 after 4 doses of 11-300 μg of antigen(see FIG. 5).

Antibody titers rose dramatically for all but the lowest dose groupfollowing the third immunization with increases in GMTs ranging from 5-to 25-fold. Low antibody responses were then detectable for even the 0.4μg recipients. The 1.2 and 3.7 μg groups had comparable titers with GMTsof about 1000 and the highest four doses clustered together with GMTs ofabout 25,000, with the exception of the 33 ug dose group with a lowerGMT of 3000. Following the fourth immunization, the titer increase wasmore modest for most groups. There was a clear dose response across thelower antigen dose groups from 0.14 μg to 11 μg ranging from nodetectable antibody for recipients of 0.14 μg to a GMT of 36,000 forrecipients of 11 μg. Again, titers for the four highest dose groups of11 to 300 μg clustered together. Thus following two immunizations, theantibody titer was dependent on the antigen dose across the broad rangefrom 0.4 to 300 μg. By the third immunization, titers of the highestfour doses were all comparable and they remained at a plateau after anadditional immunization.

One month following the fourth immunization, titers were 2- to 3-foldhigher in the 300 μg group than those measured from blood drawn fivedays following the immunization (FIG. 6). This observation suggests thatthe peak anamnestic antibody response occurred later than 5 dayspost-immunization. A more modest (50%) increase was seen at this time inthe 33 μg group. In the 300 μg dose group at two months following thelast dose, GMTs declined steeply by about 70%. After another month, thedecline was less steep at 45% (100 μg) and about 14% for the 33 and 11μg doses. Thus, the rate of decline in circulating antibody titersfollowing cessation of immunization appears to be biphasic with a steepdecline the first month following peak response followed by a moremodest rate of decrease thereafter.

The antibody titers and the kinetics of the response of these SwissWebster mice are similar to those of young heterozygous PDAPP transgenicmice immunized in a parallel manner. Dosages effective to induce animmune response in humans are typically similar to dosages effective inmice.

III. Screen for Therapeutic Efficacy Against Established AD

This assay is designed to test immunogenic agents for activity inarresting or reversing neuropathologic characteristics of Aβ in agedanimals. Immunizations with 42 amino acid long Aβ (AN1792) were begun ata time point when amyloid plaques are already present in the brains ofthe PDAPP mice.

Over the time course used in this study, untreated PDAPP mice develop anumber of neurodegenerative changes that resemble those found in Aβ(Games et al., supra and Johnson-Wood et al., Proc. Natl. Acad. Sci. USA94, 1550-1555 (1997)). The deposition of Aβ into amyloid plaques isassociated with a degenerative neuronal response consisting of aberrantaxonal and dendritic elements, called dystrophic neurites. Amyloiddeposits that are surrounded by and contain dystrophic neurites calledneuritic plaques. In both Aβ and the PDAPP mouse, dystrophic neuriteshave a distinctive globular structure, are immunoreactive with a panelof antibodies recognizing APP and cytoskeletal components, and displaycomplex subcellular degenerative changes at the ultrastructural level.These characteristics allow for disease-relevant, selective andreproducible measurements of neuritic plaque formation in the PDAPPbrains. The dystrophic neuronal component of PDAPP neuritic plaques iseasily visualized with an antibody specific for human APP (monoclonalantibody 8E5), and is readily measurable by computer-assisted imageanalysis. Therefore, in addition to measuring the effects of AN1792 onamyloid plaque formation, we monitored the effects of this treatment onthe development of neuritic dystrophy.

Astrocytes and microglia are non-neuronal cells that respond to andreflect the degree of neuronal injury. GFAP-positive astrocytes and MHCII-positive microglia are commonly observed in AD, and their activationincreases with the severity of the disease. Therefore, we also monitoredthe development of reactive astrocytosis and microgliosis in theAN1792-treated mice.

A. Materials and Methods

Forty-eight, heterozygous female PDAPP mice, 11 to 11.5 months of age,obtained from Charles River, were randomly divided into two groups: 24mice to be immunized with 100 μg of AN1792 and 24 mice to be immunizedwith PBS, each combined with Freund's adjuvant. The AN1792 and PBSgroups were again divided when they reached ˜15 months of age. At 15months of age approximately half of each group of the AN1792- andPBS-treated animals were euthanized (n=10 and 9. respectively), theremainder continued to receive immunizations until termination at ˜18months (n=9 and 12, respectively). A total of 8 animals (5 AN1792, 3PBS) died during the study. In addition to the immunized animals,one-year old (n=10), 15-month old (n=10) and 18-month old (n=10)untreated PDAPP mice were included for comparison in the ELISAs tomeasure Aβ and APP levels in the brain; the one-year old animals werealso included in the immunohistochemical analyses.

Methodology was as in Example 1 unless otherwise indicated. US Peptideslot 12 and California Peptides lot ME0339 of AN1792 were used to preparethe antigen for the six immunizations administered prior to the 15-monthtime point. California Peptides lots ME0339 and ME0439 were used for thethree additional immunizations administered between 15 and 18 months.

For immunizations, 100 μg of AN1792 in 200 μl PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) orIncomplete Freund's adjuvant (IFA) or PBS in a final volume of 400 μl.The first immunization was delivered with CFA as adjuvant, the next fourdoses were given with IFA and the final four doses with PBS alonewithout added adjuvant. A total of nine immunizations were given overthe seven-month period on a two-week schedule for the first three dosesfollowed by a four-week interval for the remaining injections. Thefour-month treatment group, euthanized at 15 months of age, receivedonly the first 6 immunizations.

B. Results

1. Effects of AN1792 Treatment on Amyloid Burden

The results of AN1792 treatment on cortical amyloid burden determined byquantitative image analysis are shown in FIG. 7. The median value ofcortical amyloid burden was 0.28% in a group of untreated 12-month oldPDAPP mice, a value representative of the plaque load in mice at thestudy's initiation. At 18 months, the amyloid burden increased over17-fold to 4.87% in PBS-treated mice, while AN1792-treated mice had agreatly reduced amyloid burden of only 0.01%, notably less than the12-month untreated and both the 15- and 18-month PBS-treated groups. Theamyloid burden was significantly reduced in the AN1792 recipients atboth 15 (96% reduction; p=0.003) and 18 (>99% reduction; p=0.0002)months.

Typically, cortical amyloid deposition in PDAPP mice initiates in thefrontal and retrosplenial cortices (RSC) and progresses in aventral-lateral direction to involve the temporal and entorhinalcortices (EC). Little or no amyloid was found in the EC of 12 month-oldmice, the approximate age at which AN1792 was first administered. After4 months of AN1792 treatment, amyloid deposition was greatly diminishedin the RSC, and the progressive involvement of the EC was entirelyeliminated by AN1792 treatment. The latter observation showed thatAN1792 completely halted the progression of amyloid that would normallyinvade the temporal and ventral cortices, as well as arrested orpossibly reversed deposition in the RSC.

The profound effects of AN1792 treatment on developing cortical amyloidburden in the PDAPP mice are further demonstrated by the 18-month group,which had been treated for seven months. A near complete absence ofcortical amyloid was found in the AN1792-treated mouse, with a totallack of diffuse plaques, as well as a reduction in compacted deposits.

2. AN1792 Treatment-associated Cellular and Morphological Changes

A population of Aβ-positive cells was found in brain regions thattypically contain amyloid deposits. Remarkably, in several brains fromAN1792 recipients, very few or no extracellular cortical amyloid plaqueswere found. Most of the Aβ immunoreactivity appeared to be containedwithin cells with large lobular or clumped soma. Phenotypically, thesecells resembled activated microglia or monocytes. They wereimmunoreactive with antibodies recognizing ligands expressed byactivated monocytes and microglia (MHC II and CD11b) and wereoccasionally associated with the wall or lumen of blood vessels.Comparison of near-adjacent sections labeled with Aβ and MHC II-specificantibodies revealed that similar patterns of these cells were recognizedby both classes of antibodies. Detailed examination of theAN1792-treated brains revealed that the MHC II-positive cells wererestricted to the vicinity of the limited amyloid remaining in theseanimals. Under the fixation conditions employed, the cells were notimmunoreactive with antibodies that recognize T cell (CD3, CD3e) or Bcell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), butwere reactive with an antibody recognizing leukosialin (CD43) whichcross-reacts with monocytes. No such cells were found in any of thePBS-treated mice.

PDAPP mice invariably develop heavy amyloid deposition in the outermolecular layer of the hippocampal dentate gyrus. The deposition forms adistinct streak within the perforant pathway, a subregion thatclassically contains amyloid plaques in AD. The characteristicappearance of these deposits in PBS-treated mice resembled thatpreviously characterized in untreated PDAPP mice. The amyloid depositionconsisted of both diffuse and compacted plaques in a continuous band. Incontrast, in a number of brains from AN1792-treated mice this patternwas drastically altered. The hippocampal amyloid deposition no longercontained diffuse amyloid, and the banded pattern was completelydisrupted. Instead, a number of unusual punctate structures were presentthat are reactive with anti-Aβ antibodies, several of which appeared tobe amyloid-containing cells.

MHC II-positive cells were frequently observed in the vicinity ofextracellular amyloid in AN1792-treated animals. The pattern ofassociation of Aβ-positive cells with amyloid was very similar inseveral brains from AN1792-treated mice. The distribution of thesemonocytic cells was restricted to the proximity of the deposited amyloidand was entirely absent from other brain regions devoid of Aβ plaques.Confocal microscopy of MHCII- and Aβ-labelled sections revealed thatplaque material was contained within many of the monocytic cells.

Quantitative image analysis of MHC II and MAC I-labeled sectionsrevealed a trend towards increased immunoreactivity in the RSC andhippocampus of AN1792-treated mice compared to the PBS group whichreached significance with the measure of MAC 1 reactivity inhippocampus.

These results are indicative of active, cell-mediated clearance ofamyloid in plaque-bearing brain regions.

3. AN1792 Effects on Aβ Levels: ELISA Determinations

(a) Cortical Levels

In untreated PDAPP mice, the median level of total Aβ in the cortex at12 months was 1,600 ng/g, which increased to 8,700 ng/g by 15 months(Table 2). At 18 months the value was 22,000 ng/g, an increase of over10-fold during the time course of the experiment. PBS-treated animalshad 8,600 ng/g total Aβ at 15 months which increased to 19,000 ng/g at18 months. In contrast, AN1792-treated animals had 81% less total Aβ at15 months (1,600 ng/g) than the PBS-immunized group. Significantly less(p=0.0001) total Aβ (5,200 ng/g) was found at 18 months when the AN11792and PBS groups were compared (Table 2), representing a 72% reduction inthe Aβ that would otherwise be present. Similar results were obtainedwhen cortical levels of Aβ42 were compared, namely that theAN1792-treated group contained much less Aβ42, but in this case thedifferences between the AN1792 and PBS groups were significant at both15 months (p=0.04) and 18 months (p=0.0001, Table 2).

TABLE 2 Median Aβ Levels (ng/g) in Cortex UNTREATED PBS AN1792 Age TotalAβ42 (n) Total Aβ42 (n) Total Aβ42 (n) 12  1,600  1,300 (10) 15  8,700 8,300 (10)  8,600  7,200  (9) 1,600   1,300*  (10) 18 22,200 18,500(10) 19,000 15,900 (12) 5,200** 4,000**  (9) *p = 0.0412 **p = 0.0001

(b) Hippocampal Levels

In untreated PDAPP mice, median hippocampal levels of total Aβ at twelvemonths of age were 15,000 ng/g which increased to 51,000 ng/g at 15months and further to 81,000 ng/g at 18 months (Table 3). Similarly, PBSimmunized mice showed values of 40,000 ng/g and 65,000 ng/g at 15 monthsand 18 months, respectively. AN1792 immunized animals exhibited lesstotal Aβ, specifically 25,000 ng/g and 51,000 ng/g at the respective15-month and 18-month timepoints. The 18-month AN1792-treated groupvalue was significantly lower than that of the PBS treated group(p=0.0105; Table 3). Measurement of Aβ42 gave the same pattern ofresults, namely that levels in the AN1792-treated group weresignificantly lower than in the PBS group (39,000 ng/g vs. 57,000 ng/g,respectively; p=0.002) at the 18-month evaluation (Table 3).

TABLE 3 Median Aβ Levels (ng/g) in Hippocampus UNTREATED PBS AN1792 AgeTotal Aβ42 (n) Total Aβ42 (n) Total Aβ42 (n) 12 15,500 11,100 (10) 1551,500 44,400 (10) 40,100 35,70  (9) 24,50 22,100   (10) 18 80,00064,200 (10) 65,400 57,10 (12) 50,90 38,900**  (9) *p = 0.0105 **p =0.0022

(c) Cerebellar Levels

In 12-month untreated PDAPP mice, the median cerebellar level of totalAβ was 15 ng/g (Table 4). At 15 months, this median increased to 28 ng/gand by 18 months had risen to 35 ng/g. PBS-treated animals displayedmedian total Aβ values of 21 ng/g at 15 months and 43 ng/g at 18 months.AN1792-treated animals were found to have 22 ng/g total Aβ at 15 monthsand significantly less (p=0.002) total Aβ at 18 months (25 ng/g) thanthe corresponding PBS group (Table 4).

TABLE 4 Median Aβ Levels (ng/g) in Cerebellum UNTREATED PBS AN1792 AgeTotal Aβ (n) Total Aβ (n) Total Aβ (n) 12 15.6 (10) 15 27.7 (10) 20.8 (9) 21.7 (10) 18 35.0 (10) 43.1 (12) 24.8*  (9) *p = 0.0018

4. Effects of AN1792 Treatment on APP Levels

APP-α and the full-length APP molecule both contain all or part of theAβ sequence and thus could be potentially impacted by the generation ofan AN1792-directed immune response. In studies to date, a slightincrease in APP levels has been noted as neuropathology increases in thePDAPP mouse. In the cortex, levels of either APP-α/FL (full length) orAPP-α were essentially unchanged by treatment with the exception thatAPP-α was reduced by 19% at the 18-month timepoint in the AN1792-treatedvs. the PBS-treated group. The 18-month AN1792-treated APP values werenot significantly different from values of the 12-month and 15-monthuntreated and 15-month PBS groups. In all cases the APP values remainedwithin the ranges that are normally found in PDAPP mice.

5. Effects of AN1792 Treatment on Neurodegenerative and GlioticPathology

Neuritic plaque burden was significantly reduced in the frontal cortexof AN1792-treated mice compared to the PBS group at both 15 (84%;p=0.⁰³) and 18 (55%; p=0.01) months of age (FIG. 8). The median value ofthe neuritic plaque burden increased from 0.32% to 0.49% in the PBSgroup between 15 and 18 months of age. This contrasted with the greatlyreduced development of neuritic plaques in the AN1792 group, with medianneuritic plaque burden values of 0.05% and 0.22%, in the 15 and 18 monthgroups, respectively.

Immunizations with AN1792 seemed well tolerated and reactiveastrocytosis was also significantly reduced in the RSC of AN1792-treatedmice when compared to the PBS group at both 15 (56%; p=0.01 1) and 18(39%; p=0.028) months of age (FIG. 9). Median values of the percent ofastrocytosis in the PBS group increased between 15 and 18 months from4.26% to 5.21%. AN1792-treatment suppressed the development ofastrocytosis at both time points to 1.89% and 3.2%, respectively. Thissuggests the neuropil was not being damaged by the clearance process.

6. Antibody Responses

As described above, eleven-month old, heterozygous PDAPP mice (N=24)received a series of 5 immunizations of 100 μg of AN1792 emulsified withFreund's adjuvant and administered intraperitoneally at weeks 0, 2, 4,8, and 12, and a sixth immunization with PBS alone (no Freund'sadjuvant) at week 16. As a negative control, a parallel set of 24age-matched transgenic mice received immunizations of PBS emulsifiedwith the same adjuvants and delivered on the same schedule. Animals werebled within three to seven days following each immunization startingafter the second dose. Antibody responses to AN1792 were measured byELISA. Geometric mean titers (GMT) for the animals that were immunizedwith AN1792 were approximately 1,900, 7,600, and 45,000 following thesecond, third and last (sixth) doses respectively. No Aβ-specificantibody was measured in control animals following the sixthimmunization.

Approximately one-half of the animals were treated for an additionalthree months, receiving immunizations at about 20, 24 and 27 weeks. Eachof these doses was delivered in PBS vehicle alone without Freund'sadjuvant. Mean antibody titers remained unchanged over this time period.In fact, antibody titers appeared to remain stable from the fourth tothe eighth bleed corresponding to a period covering the fifth to theninth injections.

To determine if the Aβ-specific antibodies elicited by immunization thatwere detected in the sera of AN1792-treated mice were also associatedwith deposited brain amyloid, a subset of sections from the AN1792- andPBS-treated mice were reacted with an antibody specific for mouse IgG.In contrast to the PBS group, Aβ plaques in AN1792-treated brains werecoated with endogenous IgG. This difference between the two groups wasseen in both 15-and 18-month groups. Particularly striking was the lackof labeling in the PBS group, despite the presence of a heavy amyloidburden in these mice. These results show that immunization with asynthetic Aβ protein generates antibodies that recognize and bind invivo to the Aβ in amyloid plaques.

7. Cellular-Mediated Immune Responses

Spleens were removed from nine AN1792-immunized and 12 PBS-immunized18-month old PDAPP mice 7 days after the ninth immunization. Splenocyteswere isolated and cultured for 72 h in the presence of Aβ40, Aβ42, orAβ40-1 (reverse order protein). The mitogen Con A served as a positivecontrol. Optimum responses were obtained with>1.7 μM protein. Cells fromall nine AN1792-treated animals proliferated in response to eitherAβ1-40 or Aβ1-42 protein, with equal levels of incorporation for bothproteins (FIG. 10A. There was no response to the Aβ40-1 reverse protein.Cells from control animals did not respond to any of the Aβ proteins(FIG. 10B).

C. Conclusion

The results of this study show that AN1792 immunization of PDAPP micepossessing existing amyloid deposits slows and prevents progressiveamyloid deposition and retard consequential neuropathologic changes inthe aged PDAPP mouse brain. Immunizations with AN1792 essentially haltedamyloid developing in structures that would normally succumb toamyloidosis. Thus, administration of Aβ peptide has therapeutic benefitin the treatment of AD.

IV. Screen of Aβ Fragments

100 PDAPP mice age 9-11 months were immunized with 9 different regionsof APP and, A to determine which epitopes convey the efficaciousresponse. The 9 different immunogens and one control are injected i.p.as described above. The immunogens include four human Aβ peptideconjugates 1-12, 13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgGvia a cystine link; an APP polypeptide amino acids 592-695, aggregatedhuman Aβ1-40, and aggregated human Aβ25-35, and aggregated rodent Aβ42.Aggregated Aβ42 and PBS were used as positive and negative controls,respectively. Ten mice were used per treatment group. Titers weremonitored as above and mice were euthanized at the end of 4 months ofinjections. Histochemistry, Aβ levels, and toxicology analysis wasdetermined post mortem.

A. Materials and Methods

1. Preparation of Immunogens

Preparation of coupled Aβ peptides: four human Aβ peptide conjugates(amino acid residues 1-5, 1-12, 13-28, and 33-42, each conjugated tosheep anti-mouse IgG) were prepared by coupling through an artificialcysteine added to the Aβ peptide using the crosslinking reagentsulfo-EMCS. The Aβ peptide derivatives were synthesized with thefollowing final amino acid sequences. In each case, the location of theinserted cysteine residue is indicated by underlining. The Aβ13-28peptide derivative also had two glycine residues added prior to thecarboxyl terminal cysteine as indicated.

Aβ1-12 peptide NH2-DAEFRHDSGYEVC-COOH (SEQ ID NO:72) Aβ1-5 peptideNH2-DAEFRC-COOH (SEQ ID NO:73) Aβ33-42 peptide NH2-C-amino-heptanoicacid-GLMVGGVVIA-COOH (SEQ ID NO:74) Aβ13-28 peptideAc-NH-HHQKLVFFAEDVGSNKGGC-COOH (SEQ ID NO:75)

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody was thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS

[N (ε-maleimidocuproyloxy) succinimide] (Molecular Sciences Co.) wasdissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS was added dropwise with stirring to the sheep anti-mouse IgGand then the solution was stirred for an additional ten min. Theactivated sheep anti-mouse IgG was purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO4, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, were pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of Aβ peptide was dissolved in 20 mL of 10 mM NaPO4, pH8.0, with the exception of the Aβ33-42 peptide for which 10 mg was firstdissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10 mMNaPO4 If buffer. The peptide solutions were each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates were concentrated to a final volume of lessthan 10 mL using an Amicon Centriprep tube and then dialyzed against PBSto buffer exchange the buffer and remove free peptide. The conjugateswere passed through 0.22 μm-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates were determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation was documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG. The Aβ1-5 sheep anti-mouse conjugate was a pool of twoconjugations, the rest were from a single preparation. 2. Preparation ofaggregated Aβ peptides

Human 1-40 (AN1528; California Peptides Inc., Lot ME0541), human 1-42(AN1792; California Peptides Inc., Lots ME0339 and ME0439), human 25-35,and rodent 1j42 (California Peptides Inc., Lot ME0218) peptides werefreshly solubilized for the preparation of each set of injections fromlyophilized powders that had been stored desiccated at −20° C. For thispurpose, two mg of peptide were added to 0.9 ml of deionized water andthe mixture was vortexed to generate a relatively uniform solution orsuspension. Of the four, AN1528 was the only peptide soluble at thisstep. A 100 μl aliquot of 10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodiumphosphate, pH 7.5) was then added at which point AN1528 began toprecipitate. The suspension was vortexed again and incubated overnightat 37° C. for use the next day.

Preparation of the pBx6 protein: An expression plasmid encoding pBx6, afusion protein consisting of the 100-amino acid bacteriophage MS-2polymerase N-terminal leader sequence followed by amino acids 592-695 ofAPP (βAPP) was constructed as described by Oltersdorf et al., J. Biol.Chem. 265, 4492-4497 (1990). The plasmid was transfected into E. coliand the protein was expressed after induction of the promoter. Thebacteria were lysed in 8M urea and pBx6 was partially purified bypreparative SDS PAGE. Fractions containing pBx6 were identified byWestern blot using a rabbit anti-pBx6 polyclonal antibody, pooled,concentrated using an Amicon Centriprep tube and dialysed against PBS.The purity of the preparation, estimated by Coomassie Blue stained SDSPAGE, was approximately 5 to 10%.

B. Results and Discussion

1. Study Design

One hundred male and female, nine- to eleven-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratory andTaconic Laboratory. The mice were sorted into ten groups to be immunizedwith different regions of Aβ or APP combined with Freund's adjuvant.Animals were distributed to match the gender, age, parentage and sourceof the animals within the groups as closely as possible. The immunogensincluded four Aβ peptides derived from the human sequence, 1-5, 1-12,13-28, and 33-42, each conjugated to sheep anti-mouse IgG; fouraggregated Aβ peptides, human 1-40 (AN1528), human 142 (AN1792), human25-35, and rodent 1-42; and a fusion polypeptide, designated as pBx6,containing APP amino acid residues 592-695. A tenth group was immunizedwith PBS combined with adjuvant as a control.

For each immunization, 100 μg of each Aβ peptide in 200 μl PBS or 200 μgof the APP derivative pBx6 in the same volume of PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) in afinal volume of 400 μl for the first immunization, followed by a boostof the same amount of immunogen in Incomplete Freund's adjuvant (IFA)for the subsequent four doses and with PBS for the final dose.Immunizations were delivered intraperitoneally on a biweekly schedulefor the first three doses, then on a monthly schedule thereafter.Animals were bled four to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.

2. Aβ and APP Levels in the Brain

Following about four months of immunization with the various Aβ peptidesor the APP derivative, brains were removed from saline-perfused animals.One hemisphere was prepared for immunohistochemical analysis and thesecond was used for the quantitation of Aβ and APP levels. To measurethe concentrations of various forms of beta amyloid peptide and amyloidprecursor protein, the hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and the level of amyloid or APP wasquantitated by comparison to a series of dilutions of standards of Aβpeptide or APP of known concentrations in an ELISA format.

The median concentration of total Aβ for the control group immunizedwith PBS was 5.8-fold higher in the hippocampus than in the cortex(median of 24,318 ng/g hippocampal tissue compared to 4,221 ng/g for thecortex). The median level in the cerebellum of the control group (23.4ng/g tissue) was about 1,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997, supra).

For the cortex, a subset of treatment groups had median total Aβ andA—1-42 levels which differed significantly from those of the controlgroup (p<0.05), those animals receiving AN1792, rodent Aβ1-42 or theAβ1-5 peptide conjugate as shown in FIG. 11. The median levels of totalAβ were reduced by 75%, 79% and 61%, respectively, compared to thecontrol for these treatment groups. There were no discernablecorrelations between Aβ-specific antibody titers and Aβ levels in thecortical region of the brain for any of the groups.

In the hippocampus, the median reduction of total Aβ associated withAN1792 treatment (46%, p=0.0543) was not as great as that observed inthe cortex (75%, p=0.0021). However, the magnitude of the reduction wasfar greater in the hippocampus than in the cortex, a net reduction of11,186 ng/g tissue in the hippocampus versus 3,171 ng/g tissue in thecortex. For groups of animals receiving rodent Aβ42 or Aβ1-5, the mediantotal Aβ levels were reduced by 36% and 26%, respectively. However,given the small group sizes and the high variability of the amyloidpeptide levels from animal to animal within both groups, thesereductions were not significant. When the levels of Aβ142 were measuredin the hippocampus, none of the treatment-induced reductions reachedsignificance. Thus, due to the smaller Aβ burden in the cortex, changesin this region are a more sensitive indicator of treatment effects. Thechanges in Aβ levels measured by ELISA in the cortex are similar, butnot identical, to the results from the immunohistochemical analysis (seebelow).

Total Aβ was also measured in the cerebellum, a region typicallyminimally affected with Aβ pathology. None of the median Aβconcentrations of any of the groups immunized with the various Aβpeptides or the APP derivative differed from that of the control groupin this region of the brain. This result suggests that non-pathologicallevels of Aβ are unaffected by treatment.

APP concentration was also determined by ELISA in the cortex andcerebellum from treated and control mice. Two different APP assays wereutilized. The first, designated APP-α/FL, recognizes both APP-alpha (α,the secreted form of APP which has been cleaved within the Aβ sequence),and full-length forms (FL) of APP, while the second recognizes onlyAPP-α. In contrast to the treatment-associated diminution of Aβ in asubset of treatment groups, the levels of APP were unchanged in all ofthe treated compared to the control animals. These results indicate thatthe immunizations with Aβ peptides are not depleting APP; rather thetreatment effect is specific to Aβ.

In summary, total Aβ and Aβ1-42 levels were significantly reduced in thecortex by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugate. Inthe hippocampus, total Aβ was significantly reduced only by AN1792treatment. No other treatment-associated changes in Aβ or APP levels inthe hippocampal, cortical or cerebellar regions were significant.

2. Histochemical Analyses

Brains from a subset of six groups were prepared for immunohistochemicalanalysis, three groups immunized with the Aβ peptide conjugates Aβ1-5,Aβ1-12, and Aβ13-28; two groups immunized with the full length Aβaggregates AN1792 and AN15228and the PBS-treated control group. Theresults of image analyses of the amyloid burden in brain sections fromthese groups are shown in FIG. 12. There were significant reductions ofamyloid burden in the cortical regions of three of the treatment groupsversus control animals. The greatest reduction of amyloid burden wasobserved in the group receiving AN1792 where the mean value was reducedby 97% (p=0.001). Significant reductions were also observed for thoseanimals treated with AN1528 (95%, p=0.005) and the Aβ1-5 peptideconjugate (67%, p=0.02).

The results obtained by quantitation of total Aβ or Aβ1-42 by ELISA andamyloid burden by image analysis differ to some extent. Treatment withAN1528 had a significant impact on the level of cortical amyloid burdenwhen measured by quantitative image analysis but not on theconcentration of total Aβ in the same region when measured by ELISA. Thedifference between these two results is likely to be due to thespecificities of the assays. Image analysis measures only insoluble Aβaggregated into plaques. In contrast, the ELISA measures all forms ofAβ, both soluble and insoluble, monomeric and aggregated. Since thedisease pathology is thought to be associated with the insolubleplaque-associated form of Aβ, the image analysis technique may have moresensitivity to reveal treatment effects. However since the ELISA is amore rapid and easier assay, it is very useful for screening purposes.Moreover it may reveal that the treatment-associated reduction of Aβ isgreater for plaque-associated than total Aβ.

To determine if the Aβ-specific antibodies elicited by immunization inthe treated animals reacted with deposited brain amyloid, a subset ofthe sections from the treated animals and the control mice were reactedwith an antibody specific for mouse IgG. In contrast to the PBS group,Aβ-containing plaques were coated with endogenous IgG for animalsimmunized with the Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; andthe full length Aβ aggregates AN1792 and AN1528. Brains from animalsimmunized with the other Aβ peptides or the APP peptide pBx6 were notanalyzed by this assay.

3. Measurement of Antibody Titers

Mice were bled four to seven days following each immunization startingafter the second immunization, for a total of five bleeds. Antibodytiters were measured as Aβ1-42-binding antibody using a sandwich ELISAwith plastic multi-well plates coated with Aβ1-42. As shown in FIG. 13,peak antibody titers were elicited following the fourth dose for thosefour immunogenic formulations which elicited the highest titers ofAN1792-specific antibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT:88,231), Aβ1-12 conjugate (peak GMT: 47,216)and rodent Apt-42 (peak GMT:10,766). Titers for these groups declined somewhat following the fifthand sixth doses. For the remaining five immunogens, peak titers werereached following the fifth or the sixth dose and these were of muchlower magnitude than those of the four highest titer groups: Aβ1-5conjugate (peak GMT: 2,356), pBx6 (peak GMT: 1,986), Aβ13-28 conjugate(peak GMT: 1,183), Aβ3342 conjugate (peak GMT: 658), Aβ25-35 (peak GMT:125). Antibody titers were also measured against the homologous peptidesusing the same ELISA sandwich format for a subset of the immunogens,those groups immunized with Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 or rodentAβ1-42. These titers were about the same as those measured againstAβ1-42 except for the rodent Aβ1-42 immunogen in which case antibodytiters against the homologous immnunogen were about two-fold higher. Themagnitude of the AN1792-specific antibody titer of individual animals orthe mean values of treatment groups did not correlate with efficacymeasured as the reduction of Aβ in the cortex.

4. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested approximately one week following the final, sixth,immunization. Freshly harvested cells, 105 per well, were cultured for 5days in the presence of Aβ1-40 at a concentration of 5 μM forstimulation. Cells from a subset of seven of the ten groups were alsocultured in the presence of the reverse peptide, Aβ40-1. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Lymphocytes from a majority of the animals proliferated in response toPHA. There were no significant responses to the Aβ40-1 reverse peptide.Cells from animals immunized with the larger aggregated Aβ peptides,AN1792, rodent Aβ1-42 and AN1528 proliferated robustly when stimulatedwith Aβ1-40 with the highest cpm in the recipients of AN1792. One animalin each of the groups immunized with Aβ1-12 conjugate, Aβ13-28 conjugateand Aβ25-35 proliferated in response to Aβ1-40. The remaining groupsreceiving Aβ1-5 conjugate, Aβ33-42 conjugate pBx6 or PBS had no animalswith an Aβ-stimulated response. These results are summarized in Table 5below.

TABLE 5 Immunogen Conjugate Aβ Amino Acids Responders Aβ1-5 Yes  5-mer0/7 Aβ1-12 Yes 12-mer 1/8 Aβ13-28 Yes 16-mer 1/9 Aβ25-35 11-mer 1/9Aβ33-42 Yes 10-mer  0/10 Aβ1-40 40-mer 5/8 Aβ1-42 42-mer 9/9 r Aβ1-4242-mer 8/8 pBx6 0/8 PBS  0-mer 0/8

These results show that AN1792 and AN1528 stimulate strong T cellresponses, most likely of the CD4+phenotype. The absence of anAβ-specific T cell response in animals immunized with Aβ1-5 is notsurprising since peptide epitopes recognized by CD4+T cells are usuallyabout 15 amino acids in length, although shorter peptides can sometimesfunction with less efficiency. Thus the majority of helper T cellepitopes for the four conjugate peptides are likely to reside in the IgGconjugate partner, not in the Aβ region. This hypothesis is supported bythe very low incidence of proliferative responses for animals in each ofthese treatment groups. Since the Aβ1-5 conjugate was effective atsignificantly reducing the level of Aβ in the brain, in the apparentabsence of Aβ-specific T cells, the key effector immune response inducedby immunization with this peptide appears to be antibody.

Lack of T-cell and low antibody response from fusion peptide pBx6,encompassing APP amino acids 592-695 including all of the Aβ residuesmay be due to the poor immunogenicity of this particular preparation.The poor immunogenicity of the Aβ25-35 aggregate is likely due to thepeptide being too small to be likely to contain a good T cell epitope tohelp the induction of an antibody response. If this peptide wereconjugated to a carrier protein, it would probably be more immunogenic.

V. Preparation of Polyclonal Antibodies for Passive Protection

125 non-transgenic mice were immunized with 100 μg Aβ1-42, plus CFA/IFAadjuvant, and euthanized at 4-5 months. Blood was collected fromimmunized mice. IgG was separated from other blood components. Antibodyspecific for the immunogen may be partially purified by affinitychromatography. An average of about 0.5-1 mg of immunogen-specificantibody is obtained per mouse, giving a total of 60-120 mg.

VI. Passive Immunization with Antibodies to Aβ

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-β monoclonals as shown below. Thecell line designated RB44-10D5.19.21 producing the antibody 10D5 has theATCC accession number PTA-5129, having been deposited on Apr. 8. 2003.All antibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

TABLE 6 Antibody Epitope 2H3 Aβ1-12 10D5 Aβ1-12 266 Aβ13-28 21F12Aβ33-42 Mouse polyclonal Anti-Aggregated Aβ42 anti-human Aβ42

Mice were injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1/1000 defined by ELISA to Aβ42 or other immunogen. Titers weremonitored as above and mice were euthanized at the end of 6 months ofinjections. Histochemistry, Aβ levels and toxicology were performed postmortem. Ten mice were used per group. Additional studies of passiveimmunization are described in Examples XI and XII below.

VII. Comparison of Different Adjuvants

This example compares CFA, alum, an oil-in water emulsion and MPL forcapacity to stimulate an immune response.

A. Materials and Methods

1. Study Design

One hundred female Hartley strain six-week old guinea pigs, obtainedfrom Elm Hill, were sorted into ten groups to be immunized with AN1792or a palmitoylated derivative thereof combined with various adjuvants.Seven groups received injections of AN1792 (33 μg unless otherwisespecified) combined with a) PBS, b) Freund's adjuvant, c) MPL, d)squalene, e) MPL/squalene f) low dose alum, or g) high dose alum (300 μgAN1792). Two groups received injections of a palmitoylated derivative ofAN1792 (33 μg) combined with a) PBS or b) squalene. A final, tenth groupreceived PBS alone without antigen or additional adjuvant. For the groupreceiving Freund's adjuvant, the first dose was emulsified with CFA andthe remaining four doses with IFA. Antigen was administered at a dose of33 μg for all groups except the high dose alum group, which received 300μg of AN1792. Injections were administered intraperitoneally for CFA/IFAand intramuscularly in the hind limb quadriceps alternately on the rightand left side for all other groups. The first three doses were given ona biweekly schedule followed by two doses at a monthly interval). Bloodwas drawn six to seven days following each immunization, starting afterthe second dose, for measurement of antibody titers.

2. Preparation of Immunogens

Two mg Aβ42 (California Peptide, Lot ME0339) was added to 0.9 ml ofdeionized water and the mixture was vortexed to generate a relativelyuniform suspension. A 100 μl aliquot of 10×PBS (1×PBS, 0.15 M NaCl, 0.01M sodium phosphate, pH 7.5) was added. The suspension was vortexed againand incubated overnight at 37° C. for use the next day. Unused Aβ142 wasstored with desiccant as a lyophilized powder at −20° C.

A palmitoylated derivative of AN1792 was prepared by coupling palmiticanhydride, dissolved in dimethyl formamide, to the amino terminalresidue of AN1792 prior to removal of the nascent peptide from the resinby treatment with hydrofluoric acid.

To prepare formulation doses with Complete Freund's adjuvant (CFA)(group 2), 33 μg of AN1792 in 200 μl PBS was emulsified 1:1 (vol:vol)with CFA in a final volume of 400 μl for the first immunization. Forsubsequent immunizations, the antigen was similarly emulsified withIncomplete Freund's adjuvant (IFA).

To prepare formulation doses with MPL for groups 5 and 8, lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.) was added to0.2% aqueous triethylamine to a final concentration of 1 mg/ml andvortexed. The mixture was heated to 65 to 70° C. for 30 sec to create aslightly opaque uniform suspension of micelles. The solution was freshlyprepared for each set of injections. For each injection in group 5, 33μg of AN1792 in 16.5 μl PBS, 50 μg of MPL (50 μl) and 162 μl of PBS weremixed in a borosilicate tube immediately before use.

To prepare formulation doses with the low oil-in-water emulsion, AN1792in PBS was added to 5% squalene, 0.5% Tween 80, 0.5% Span 85 in PBS toreach a final single dose concentration of 33 μg AN1792 in 250 pi (group6). The mixture was emulsified by passing through a two-chamberedhand-held device 15 to 20 times until the emulsion droplets appeared tobe about equal in diameter to a 1.0 μm diameter standard latex bead whenviewed under a microscope. The resulting suspension was opalescent,milky white. The emulsions were freshly prepared for each series ofinjections. For group 8, MPL in 0.2% triethylamine was added at aconcentration of 50 μg per dose to the squalene and detergent mixturefor emulsification as noted above. For the palmitoyl derivative (group7), 33 μg per dose of palmitoyl-NH-Aβ1-42 was added to squalene andvortexed. Tween 80 and Span 85 were then added with vortexing. Thismixture was added to PBS to reach final concentrations of 5% squalene,0.5% Tween 80, 0.5% Span 85 and the mixture was emulsified as notedabove.

To prepare formulation doses with alum (groups 9 and 10), AN1792 in PBSwas added to Alhydrogel (aluminum hydroxide gel, Accurate, Westbury,N.Y.) to reach concentrations of 33 μg (low dose, group 9) or 300 μg(high dose, group 10). AN1792 per 5 mg of alum in a final dose volume of250 μl. The suspension was gently mixed for 4 hr at RT.

3. Measurement of Antibody Titers

Guinea pigs were bled six to seven days following immunization startingafter the second immunization for a total of four bleeds. Antibodytiters against Aβ42 were measured by ELISA as described in GeneralMaterials and Methods.

4. Tissue Preparation

After about 14 weeks, all guinea pigs were euthanized by administeringC02. Cerebrospinal fluid was collected and the brains were removed andthree brain regions (hippocampus, cortex and cerebellum) were dissectedand used to measure the concentration of total Aβ protein using ELISA.

B. Results

1. Antibody Responses

There was a wide range in the potency of the various adjuvants whenmeasured as the antibody response to AN1792 following immunization. Asshown in FIG. 14, when AN1792 was administered in PBS, no antibody wasdetected following two or three immunizations and negligible responseswere detected following the fourth and fifth doses with geometric meantiters (GMTs) of only about 45. The o/w emulsion induced modest titersfollowing the third dose (GMT 255) that were maintained following thefourth dose (GMT 301) and fell with the final dose (GMT 54). There was aclear antigen dose response for AN1792 bound to alum with 300 μg beingmore immunogenic at all time points than 33 μg. At the peak of theantibody response, following the fourth immunization, the differencebetween the two doses was 43% with GMTs of about 1940 (33 μg) and 3400(300 μg). The antibody response to 33 μg AN1792 plus MPL was verysimilar to that generated with almost a ten-fold higher dose of antigen(300 μg) bound to alum. The addition of MPL to an o/w emulsion decreasedthe potency of the formulations relative to that with MPL as the soleadjuvant by as much as 75%. A palmitoylated derivative of AN1792 wascompletely non-immunogenic when administered in PBS and gave modesttiters when presented in an o/w emulsion with GMTs of 340 and 105 forthe third and fourth bleeds. The highest antibody titers were generatedwith Freund's adjuvant with a peak GMT of about 87,000, a value almost30-fold greater than the GMTs of the next two most potent formulations,MPL and high dose AN1792/alum.

The most promising adjuvants identified in this study are MPL and alum.Of these two, MPL appears preferable because a 10-fold lower antigendose was required to generate the same antibody response as obtainedwith alum. The response can be increased by increasing the dose ofantigen and /or adjuvant and by optimizing the immunization schedule.The o/w emulsion was a very weak adjuvant for AN1792 and adding an o/wemulsion to MPL adjuvant diminished the intrinsic adjuvant activity ofMPL alone.

2. Aβ Levels In The Brain

At about 14 weeks the guinea pigs were deeply anesthetized, thecerebrospinal fluid (CSF) was drawn and brains were excised from animalsin a subset of the groups, those immunized with Freund's adjuvant (group2), MPL (group 5), alum with a high dose, 300 μg, of AN1792 (group 10)and the PBS immunized control group (group 3). To measure the level ofAβ peptide, one hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and quantitated by comparison to a seriesof dilutions of Aβ standard protein of known concentrations in an ELISAformat. The levels of Aβ protein in the hippocampus, the cortex and thecerebellum were very similar for all four groups despite the wide rangeof antibody responses to Aβ elicited by these formulations. Mean Aβlevels of about 25 ng/g tissue were measured in the hippocampus, 21 ng/gin the cortex, and 12 ng/g in the cerebellum. Thus, the presence of ahigh circulating antibody titer to Aβ for almost three months in some ofthese animals did not alter the total Aβ levels in their brains. Thelevels of Aβ in the CSF were also quite similar between the groups. Thelack of large effect of AN1792 immunization on endogenous Aβ indicatesthat the immune response is focused on pathological formations of Aβ.

VIII. Immune Response to Different Adjuvants in Mice

Six-week old female Swiss Webster mice were used for this study with10-13 animals per group. Immunizations were given on days 0, 14, 28, 60,90 and 20 administered subcutaneously in a dose volume of 200 μl. PBSwas used as the buffer for all formulations. Animals were bleed sevendays following each immunization starting after the second dose foranalysis of antibody titers by ELISA. The treatment regime of each groupis summarized in Table 7.

TABLE 7 Experimental Design Dose Group N^(a) Adjuvant^(b) Dose Antigen(μg) 1 10 MPL 12.5 μg AN1792 33 2 10 MPL   25 μg AN1792 33 3 10 MPL   50μg AN1792 33 4 13 MPL  125 μg AN1792 33 5 13 MPL   50 μg AN1792 150 6 13MPL   50 μg AN1528 33 7 10 PBS AN1792 33 8 10 PBS None 9 10 Squalene 5%AN1792 33 emulsified 10 10 Squalene 5% AN1792 33 admixed 11 10 Alum 2 mgAN1792 33 12 13 MPL + Alum 50 μg/2 mg AN1792 33 13 10 QS-21  5 μg AN179233 14 10 QS-21 10 μg AN1792 33 15 10 QS-21 25 AN1792 AN1792 33 16 13QS-21 25 AN1792 AN1792 150 17 13 QS-21 25 AN1792 AN1528 33 18 13 QS-21 +MPL 25 μg/50 μg AN1792 33 19 13 QS-21 + 25 μg/2 mg AN1792 33 AlumFootnotes: ^(a)Number of mice in each group at the initiation of theexperiment. ^(b)The adjuvants are noted. The buffer for all theseformulations was PBS. For group 8, there was no adjuvant and no antigen.

The ELISA titers of antibodies against Aβ42 in each group are shown inbelow.

TABLE 8 Geometric Mean Antibody Titers Week of Bleed Treatment Group 2.95.0 8.7 12.9 16.7 1 248 1797 2577 6180 4177 2 598 3114 3984 5287 6878 31372 5000 7159 12333 12781 4 1278 20791 14368 20097 25631 5 3288 2624213229 9315 23742 6 61 2536 2301 1442 4504 7 37 395 484 972 2149 8 25 2525 25 25 9 25 183 744 952 1823 10 25 89 311 513 817 11 29 708 2618 21653666 12 198 1458 1079 612 797 13 38 433 566 1080 626 14 104 541 32471609 838 15 212 2630 2472 1224 1496 16 183 2616 6680 2085 1631 17 28 201375 222 1540 18 31699 15544 23095 6412 9059 19 63 243 554 299 441

The table shows that the highest titers were obtained for groups 4, 5and 18, in which the adjuvants were 125 μg MPL, 50 μg MPL and QS-21 plusMPL.

IX. Therapeutic Efficacy of Different Adjuvants

A therapeutic efficacy study was conducted in PDAPP transgenic mice witha set of adjuvants suitable for use in humans to determine their abilityto potentiate immune responses to Aβ and to induce the immune-mediatedclearance of amyloid deposits in the brain.

One hundred eighty male and female, 7.5- to 8.5-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratories. Themice were stored into nine groups containing 15 to 23 animals per groupto be immunized with AN1792 or AN1528 combined with various adjuvants.Animals were distributed to match the gender, age, and parentage of theanimals within the groups as closely as possible. The adjuvants includedalum, MPL, and QS-21, each combined with both antigens, and Freund'sadjuvant (FA) combined with only AN1792. An additional group wasimmunized with AN1792 formulated in PBS buffer plus the preservativethimerosal without adjuvant. A ninth group was immunized with PBS aloneas a negative control.

Preparation of aggregated Aβ peptides: human Aβ1-40 (AN1528; CaliforniaPeptides Inc., Napa, Calif.; Lot ME0541) and human Aβ1-42 (AN1792;California Peptides Inc., Lot ME0439) peptides were freshly solubilizedfor the preparation of each set of injections from lyophilized powdersthat had been stored desiccated at −20° C. For this purpose, two mg ofpeptide were added to 0.9 ml of deionized water and the mixture wasvortexed to generate a relatively uniform solution or suspension. AN1528was soluble at this step, in contrast to AN1792. A 100 μl aliquot of10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was thenadded at which point AN1528 began to precipitate. The suspensions werevortexed again and incubated overnight at 37° C. for use the next day.

To prepare formulation doses with alum (Groups 1 and 5). Aβ peptide inPBS was added to Alhydrogel (two percent aqueous aluminum hydroxide gel,Sargeant, Inc., Clifton, N.J.) to reach concentrations of 100 μg Aβpeptide per 2 mg of alum. 10×PBS was added to a final dose volume of 200ml in 1×PBS. The suspension was then gently mixed for approximately 4 hrat RT prior to injection.

To prepare formulation doses for with MPL (Groups 2 and 6), lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.; Lot67039-E0896B) was added to 0.2% aqueous triethylamine to a finalconcentration of 1 mg/ml and vortexed. The mixture was heated to 65 to70° C. for 30 sec to create a slightly opaque uniform suspension ofmicelles. The solution was stored at 4° C. For each set of injections,100 μg of peptide per dose in 50 μl PBS, 50 μg of MPL per dose (50 pi)and 100 μl of PBS per dose were mixed in a borosilicate tube immediatelybefore use.

To prepare formulation doses with QS-21 (Groups 3 and 7), lyophilizedpowder (Aquila, Framingham, MA; Lot A7018R) was added to PBS, pH 6.6-6.7to a final concentration of 1 mg/ml and vortexed. The solution wasstored at −20° C. For each set of injections, 100 μg of peptide per dosein 50 μl PBS, 25 μg of QS-21 per dose in 25 μl PBS and 125 μl of PBS perdose were mixed in a borosilicate tube immediately before use.

To prepare formulation doses with Freund's Adjuvant (Group 4), 100 g ofAN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with Complete Freund'sAdjuvant (CFA) in a final volume of 400 μl for the first immunization.For subsequent immunizations, the antigen was similarly emulsified withIncomplete Freund's Adjuvant (IFA). For the formulations containing theadjuvants alum, MPL or QS21, 100 g per dose of AN1792 or AN1528 wascombined with alum (2 mg per dose) or MPL (50 g per dose) or QS21 (25 gper dose) in a final volume of 200 μl PBS and delivered by subcutaneousinoculation on the back between the shoulder blades. For the groupreceiving FA, 100 g of AN1792 was emulsified 1:1 (vol:vol) with CompleteFreund's adjuvant (CFA) in a final volume of 400 μl and deliveredintraperitoneally for the first immunization, followed by a boost of thesame amount of immunogen in Incomplete Freund's adjuvant (IFA) for thesubsequent five doses. For the group receiving AN1792 without adjuvant,10 g AN1792 was combined with 5 g thimerosal in a final volume of 50 μlPBS and delivered subcutaneously. The ninth, control group received only200 PBS delivered subcutaneously. Immunizations were given on a biweeklyschedule for the first three doses, then on a monthly schedulethereafter on days 0, 16, 28, 56, 85 and 112. Animals were bled six toseven days following each immunization starting after the second dosefor the measurement of antibody titers. Animals were euthanizedapproximately one week after the final dose. Outcomes were measured byELISA assay of Aβ and APP levels in brain and by immunohistochemicalevaluation of the presence of amyloid plaques in brain sections. Inaddition, Aβ-specific antibody titers, and Aβ-dependent proliferativeand cytokine responses were determined.

Table 9 shows that the highest antibody titers to Aβ1-42 were elicitedwith FA and AN1792, titers which peaked following the fourthimmunization (peak GMT: 75,386) and then declined by 59% after thefinal, sixth immunization. The peak mean titer elicited by MPL withAN1792 was 62% lower than that generated with FA (peak GMT: 28,867) andwas also reached early in the immunization scheme, after 3 doses,followed by a decline to 28% of the peak value after the sixthimmunization. The peak mean titer generated with QS-21 combined withAN1792 (GMT: 1,511) was about 5-fold lower than obtained with MPL. Inaddition, the kinetics of the response were slower, since an additionalimmunization was required to reach the peak response. Titers generatedby alum-bound AN1792 were marginally greater than those obtained withQS-21 and the response kinetics were more rapid. For AN1792 delivered inPBS with thimerosal the frequency and size of titers were barely greaterthan that for PBS alone. The peak titers generated with MPL and AN1528(peak GMT 3099) were about 9-fold lower than those with AN1792.Alum-bound AN1528 was very poorly immunogenic with low titers generatedin only some of the animals. No antibody responses were observed in thecontrol animals immunized with PBS alone.

TABLE 9 Geometric Mean Antibody Titers^(a) Week of Bleed Treatment 3.35.0 9.0 13.0 17.0 Alum/ 102 1,081 2,366 1,083 572 AN1792  (12/21)^(b)(17/20) (21/21) (19/21) (18/21) MPL/ 6241 28,867 1,1242 5,665 8,204AN1792 (21/21) (21/21) (21/21) (20/20) (20/20) QS-21/ 30 227 327 1,5111,188 AN1792  (1/20) (10/19) (10/19) (17/18) (14/18) CFA/ 10,076 61,27975,386 41,628 30,574 AN1792 (15/15) (15/15) (15/15) (15/15) (15/15)Alum/ 25 33 39 37 31 AN1528  (0/21)  (1/21)  (3/20)  (1/20)  (2/20) MPL/184 2,591 1,653 1,156 3,099 AN1528 (15/21) (20/21) (21/21) (20/20)(20/20) QS-21/ 29 221 51 820 2,994 AN1528  (1/22) (13/22)  (4/22)(20/22) (21/22) PBS plus 25 33 39 37 47 Thimerosal  (0/16)  (2/16) (4/16)  (3/16)  (4/16) PBS 25 25 25 25 25  (0/16)  (0/16)  (0/15) (0/12)  (0/16) Footnotes: ^(a)Geometric mean antibody titers measuredagainst Aβ1-42 ^(b)Number of responders per group

The results of AN1792 or AN1592 treatment with various adjuvants, orthimerosal on cortical amyloid burden in 12-month old mice determined byELISA are shown in FIGS. 15A-15E. In PBS control PDAPP mice the medianlevel of total Aβ in the cortex at 12 months was 1,817 ng/g (FIG. 15A).Notably reduced levels of Aβ were observed in mice treated with AN1792plus CFA/IFA (FIG. 15C), AN1792 plus alum (FIG. 15D), AN1792 plus MPL(FIG. 15E) and QS21 plus AN1792 (FIG. 15E). The reduction reachedstatistical significance (p<0.05) only for AN1792 plus CFA/IFA (FIG.15C). However, as shown in Examples I and III, the effects ofimmunization in reducing Aβ levels become substantially greater in 15month and: 18 month old mice. Thus, it is expected that at least theAN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21 compositions willachieve statistical significance in treatment of older mice. Bycontrast, the AN1792 plus the preservative thimerosal (FIG. 15D) showeda median level of A,8 about the same as that in the PBS treated mice.Similar results were obtained when cortical levels of Aβ42 werecompared. The median level of Aβ42 in PBS controls was 1624 ng/g.Notably reduced median levels of 403, 1149, 620 and 714 were observed inthe mice treated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plusMPL and AN1792 plus QS21 respectively, with the reduction achievingstatistical significance (p=0.05) for the AN1792 CFA/IFAβ treatmentgroup. The median level in the AN1792 thimerosal treated mice was 1619ng/g A,342.

A further therapeutic adjuvant/immunogen efficacy study was performed in9-10.5 month old male and female heterozygous PDAPP transgenic mice. Theduration of the study was 25 weeks with 29-40 animals per treatmentgroup; therefore the animals were 15-16.5 months old at termination. Thetreatment groups are identified in Table 10 below.

Dilution Adjuvant Immunogen Buffer Administration Group 1: MPL-SEAN1792-GCS (75 μg) PBS SC (250 μl) Group 2: ISA 51 AN1792-GCS (75 μg)PBS IP (400 μl) Group 3: QS21 AN1792-GCS (75 μg) PBS SC (250 μl) Group4: QS21 AN1792-GCS (75 μg) PBS SC (250 μl) abbrev. Group 5: PBS — — SC(250 μl) Table 10 abbreviations: MAP - multi-antigenic peptide; TT -tetanus toxoid t-cell epitope (830-844); SQ - subcutaneous; IP -intraperitoneally; PBS - phosphate, buffered saline; ISA-51 is acommercially available adjuvant similar to IFA; GCS is aglycine/citrate/sucrose formulation, MPL-SE is MPL in a stabilized waterand oil emulsion.

The immunization schedule was identical for all of the treatment groupsexcept for Group 3, the QS21/AN1792 abbreviated schedule group. The micewere injected on weeks 0, 2, 4, 8, 12, 16, 20, 24, with bleeds on weeks3, 5, 9, 13, 17, 21 and 25. Groups 1, 2, received eight injections andGroup 3 received four injections during the 25-week period of the study.Group 4, the QS21/AN1792 abbreviated schedule, received injections onweeks 0, 2, 4, and 8 only. This group was not injected for the remainderof the study, although they were bled on the same bleed schedule as therest of the study to follow titer decay. Groups 3 and 5, QS21/AN1792 andPBS respectively, served as the positive and negative controls for thisstudy.

The titers were determined by the anti-Aβ antibody titer assay.

Group 1, the MPL-SE/AN1792 group, raised a peak geometric mean titer(GMT) of 17,100 at 9 weeks falling to a GMT of 10,000 at 25 weeks.Initially, the MPL-SE titers rose at a somewhat higher rate than theQS21/AN1792 control group (Group 4).

Group 2, the ISA 51/AN1792 group, produced high titers throughout thestudy reaching a GMT of over 100,000 for the last 9 weeks of the study.

Group 3, the QS21/AN1792 control group, reached its peak titer at 17weeks with a GMT of 16,000. The titer then fell over the next 8 weeks tofinish with a GMT of 8,700. One animal in this group failed to raise atiter over the entire course of the experiment.

Group 4, the QS21/AN1792 abbreviated injection schedule group, reached apeak titer of 7,300 at 13 weeks, five weeks after its final injection.The titer then fell to a GMT of 2,100 at the final bleed (25 weeks). Asin the control group, one animal failed to raise a detectable titer,while another animal lost all titer by the end of the decay period.

Group 5, the PBS alone group, had no titers.

To evaluate the cortical Aβ levels, total Aβ and Aβ1-42 were measured byELISA. Briefly, one brain hemisphere was dissected for cortical,hippocampal, and cerebellar tissue followed by homogenization in 5Mguanidine buffer and assayed for brain Aβ. The cortical total Aβ andAβ42 results are similar. A Mann-Whitney statistical analysis wasperformed to determine significance between the groups with a p value of0.05 indicating a significant change in Aβ.

All treatment groups significantly lowered total Aβ levels as comparedto the PBS control group (see Table 11). The MPL-SE/AN1792 group, showedthe greatest change in AB, and it is significantly better than the othertreatment groups. The QS21/AN1792 abbreviated group, was similar in itsoverall change of Aβ to the QS21 control group that received all eightinjections. The Aβ levels in the ISA 51/AN1792 group, were similarlylowered compared to the CFA/IFA:MAP(Aβ1-7) group.

TABLE 11 Cortical Aβ levels PBS MPL-SE ISA QS-21 QS-21 (4) MEDIAN 7,3351,236 3,026 2,389 2,996 (ng/g tissue) RANGE 550- 70- 23- 210- 24- (ng/gtissue) 18,358 3,977 9,777 11,167 16,834 p value — <0.0001 <0.0001<0.0001 <0.0001 N 38 29 36 34 40

In conclusion, MPL-SE, ISA-51 and QS21 adjuvants combined with AN1792are effective in inducing a sufficient immune response significantly toretard Aβ deposition in the cortex.

X. Toxicity Analysis

Tissues were collected for histopathologic examination at thetermination of studies described in Examples 2, 3 and 7. In addition,hematology and clinical chemistry were performed on terminal bloodsamples from Examples 3 and 7. Most of the major organs were evaluated,including brain, pulmonary, lymphoid, gastrointestinal, liver, kidney,adrenal and gonads. Although sporadic lesions were observed in the studyanimals, there were no obvious differences, either in tissues affectedor lesion severity, between AN1792 treated and untreated animals. Therewere no unique histopathological lesions noted in AN-1528-immunizedanimals compared to PBS-treated or untreated animals. There were also nodifferences in the clinical chemistry profile between adjuvant groupsand the PBS treated animals in Example 7. Although there weresignificant increases in several of the hematology parameters betweenanimals treated with AN1792 and Freund's adjuvant in Example 7 relativeto PBS treated animals, these type of effects are expected from Freund'sadjuvant treatment and the accompanying peritonitis and do not indicateany adverse effects from AN1792 treatment. Although not part of thetoxicological evaluation, PDAPP mouse brain pathology was extensivelyexamined as part of the efficacy endpoints. No sign of treatment relatedadverse effect on brain morphology was noted in any of the studies.These results indicate that AN1792 treatment is well tolerated and atleast substantially free of side effects.

XI. Therapeutic Treatment With Anti-Aβ Antibodies

This examples tests the capacity of various monoclonal and polyclonalantibodies to Aβ to inhibit accumulation of Aβ in the brain ofheterozygotic transgenic mice.

1. Study Design

Sixty male and female, heterozygous PDAPP transgenic mice, 8.5 to 10.5months of age were obtained from Charles River Laboratory. The mice weresorted into six groups to be treated with various antibodies directed toAll. Animals were distributed to match the gender, age, parentage andsource of the animals within the groups as closely as possible. As shownin Table 10, the antibodies included four murine A,6-specific monoclonalantibodies, 2H3 (directed to Aβ residues 1-12), 10D5 (directed to Allresidues 1-16) (details of the deposit of 10D5 are discussed in ExampleVI. suvra), 266 (directed to A, residues 13-28 and binds to monomericbut not to aggregated AN1792), 21F12 (directed to A,B residues 33-42). Afifth group was treated, with an A-specific polyclonal antibody fraction(raised by immunization with aggregated AN1792). The negative controlgroup received the diluent, PBS, alone without antibody.

The monoclonal antibodies were injected at a dose of about 10 mg/kg(assuming that the mice weighed 50 g). Injections were administeredintraperitoneally every seven days on average to maintain anti-Aβ titersabove 1000. Although lower titers were measured for mAb 266 since itdoes not bind well to the aggregated AN1792 used as the capture antigenin the assay, the same dosing schedule was maintained for this group.The group receiving monoclonal antibody 2H3 was discontinued within thefirst three weeks since the antibody was cleared too rapidly in vivo.Animals were bled prior to each dosing for the measurement of antibodytiters. Treatment was continued over a six-month period for a total of196 days. Animals were euthanized one week after the final dose.

TABLE 12 EXPERIMENTAL DESIGN Treatment Treatment Antibody Antibody GroupN^(a) Antibody Specificity Isotype 1 9 none NA^(b) NA (PBS alone) 2 10Polyclonal Aβ1-42 mixed 3 0 mAb^(c)2H3 Aβ1-12 IgG1 4 8 mAb 10D5 Aβ1-16IgG1 5 6 mAb 266 Aβ13-28 IgG1 6 8 mAb 21F12 Aβ33-42 IgG2a Footnotes^(a). Number of mice in group at termination of the experiment. Allgroups started with 10 animals per group. ^(b). NA: not applicable ^(c).mAb: monoclonal antibody

2. Materials and Methods

a. Preparation of the Antibodies

The anti-Aβ polyclonal antibody was prepared from blood collected fromtwo groups of animals. The first group consisted of 100 female SwissWebster mice, 6 to 8 weeks of age. They were immunized on days 0, 15,and 29 with 100 μg of AN1792 combined with CFA/IFA. A fourth injectionwas given on day 36 with one-half the dose of AN1792. Animals wereexsanguinated upon sacrifice at day 42, serum was prepared and the serawere pooled to create a total of 64 ml. The second group consisted of 24female mice isogenic with the PDAPP mice but nontransgenic for the humanAPP gene, 6 to 9 weeks of age. They were immunized on days 0, 14, 28 and56 with 100 μg of AN1792 combined with CFA/IFA. These animals were alsoexsanguinated upon sacrifice at day 63, serum was prepared and pooledfor a total of 14 ml. The two lots of sera were pooled. The antibodyfraction was purified using two sequential rounds of precipitation with50% saturated ammonium sulfate. The final precipitate was dialyzedagainst PBS and tested for endotoxin. The level of endotoxin was lessthan 1 EU/mg.

The anti-Aβ monoclonal antibodies were prepared from ascites fluid. Thefluid was first delipidated by the addition of concentrated sodiumdextran sulfate to ice-cold ascites fluid by stirring on ice to a reacha final concentration of 0.238%. Concentrated CaCl₂ was then added withstirring to reach a final concentration of 64 mM. This solution wascentrifuged at 10,000 ×g and the pellet was discarded. The supernatantwas stirred on ice with an equal volume of saturated ammonium sulfateadded dropwise. The solution was centrifuged again at 10,000 ×g and thesupernatant was discarded. The pellet was resuspended and dialyzedagainst 20 mM Tris-HCl , 0.4 M NaCl, pH 7.5. This fraction was appliedto a Pharmacia FPLC Sepharose Q Column and eluted with a reversegradient from 0.4 M to 0.275 M NaCl in 20 mM Tris-HCl, pH 7.5.

The antibody peak was identified by absorbance at 280 nm and appropriatefractions were pooled. The purified antibody preparation wascharacterized by measuring the protein concentration using the BCAmethod and the purity using SDS-PAGE. The pool was also tested forendotoxin. The level of endotoxin was less than 1 EU/mg. titers, titersless than 100 were arbitrarily assigned a titer value of 25.

3. Aβ and APP Levels in the Brain

Following about six months of treatment with the various anti-Aβantibody preparations, brains were removed from the animals followingsaline perfusion. One hemisphere was prepared for immunohistochemicalanalysis and the second was used for the quantitation of Aβ and APPlevels. To measure the concentrations of various forms of beta amyloidpeptide and amyloid precursor protein (APP), the hemisphere wasdissected and homogenates of the hippocampal, cortical, and cerebellarregions were prepared in 5 M guanidine. These were serially diluted andthe level of amyloid peptide or APP was quantitated by comparison to aseries of dilutions of standards of Aβ peptide or APP of knownconcentrations in an ELISA format.

The levels of total Aβ and of Aβ1-42 measured by ELISA in homogenates ofthe cortex, and the hippocampus and the level of total Aβ in thecerebellum are shown in Tables 11, 12, and 13, respectively. The medianconcentration of total Aβ for the control group, inoculated with PBS,was 3.6-fold higher in the hippocampus than in the cortex (median of63,389 ng/g hippocampal tissue compared to 17,818 ng/g for the cortex).The median level in the cerebellum of the control group (30.6 ng/gtissue) was more than 2,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Wood et al.,1997).

For the cortex, one treatment group had a median Aβ level, measured asAβ1-42, which differed significantly from that of the control group(p<0.05), those animals receiving the polyclonal anti-Aβ antibody asshown in Table 13. The median level of Aβ1-42 was reduced by 65%,compared to the control for this treatment group. The median levels ofAβ1-42 were also significantly reduced by 55% compared to the control inone additional treatment group, those animals dosed with the mAb 10D5(p=0.0433).

TABLE 13 CORTEX Medians Means Total Aβ Aβ42 Total Aβ Aβ42 TreatmentGroup N^(a) LISA value^(b) P value^(c) % Change ELISA value P value %Change ELISA value ELISA value PBS 9 17318 NA^(d) NA 13802 NA NA 16150+/− 7456^(e) 12621 +/− 5738  Polyclonal anti-Aβ42 10 6160 0.0055 −654892 0.0071 −65 5912 +/− 4492 4454 +/− 3347 mAb 10D5 8 7915 0.1019 −566214 0.0433 −55 9695 +/− 6929 6943 +/− 3351 mAb 266 6 9144 0.1255 −495481 0.1255 −39 9204 +/− 9293 7489 +/− 6921 mAb 21F12 8 15158 0.2898 −1513578 0.7003 −2 12481 +/− 7082  11005 +/− 6324  Footnotes: ^(a)Number ofanimals per group at the end of the experiment ^(b)ng/g tissue ^(c)MannWhitney analysis ^(d)NA: not applicable ^(e)Standard Deviation

In the hippocampus, the median percent reduction of total Aβ associatedwith treatment with polyclonal anti-Aβ antibody (50%, p=0.0055) was notas great as that observed in the cortex (65%) (Table 14). However, theabsolute magnitude of the reduction was almost 3-fold greater in thehippocampus than in the cortex, a net reduction of 31,683 ng/g tissue inthe hippocampus versus 11,658 ng/g tissue in the cortex. When measuredas the level of the more amyloidogenic form of Aβ, Aβ1-42, rather thanas total Aβ, the reduction achieved with the polyclonal antibody wassignificant (p=0.0025). The median levels in groups treated with themAbs 10D5 and 266 were reduced by 33% and 21%, respectively.

TABLE 14 HIPPOCAMPUS Medians Means Total Aβ Aβ42 Total Aβ Aβ42 TreatmentGroup N^(a) ELISA value^(b) P value^(c) % Change ELISA value P value %Change ELISA value ELISA value PBS 9 63389 NA^(d) NA 54429 NA NA   58351+/− 13308^(e) 52801 +/− 14701 Polyclonal anti- 10 31706 0.0055 −50 271270.0025 −50 30058 +/− 22454 24853 +/− 18262 Aβ42 mAb 10D5 8 46779 0.0675−26 36290 0.0543 −33 44581 +/− 18632 36465 +/− 17146 mAb 266 6 48689 00990 −23 43034 0.0990 −21 36419 +/− 27304 32919 +/− 25372 mAb 21F12 851563 0.7728 −19 47961 0.8099 −12 57327 +/− 28927 50305 +/− 23927Footnotes: ^(a)Number of animals per group at the end of the experiment^(b)ng/g tissue ^(c)Mann Whitney analysis ^(d)NA: not applicable^(e)Standard Deviation

Total Aβ was also measured in the cerebellum (Table 15). Those groupsdosed with the polyclonal anti-Aβ and the 266 antibody showedsignificant reductions of the levels of total Aβ (43% and 46%, p=0.0033and p=0.0184, respectively) and that group treated with 10D5 had a nearsignificant reduction (29%, p=0.0675).

TABLE 15 CEREBELLUM Medians Total Aβ Means Treatment ELISA P % Total AβGroup N^(a) value^(b) value^(c) Change ELISA value PBS 9 30.64 NA^(d) NA40.00 +/− 31.89^(e) Polyclonal 10 17.61 0.0033 −43 18.15 +/− 4.36anti-Aβ42 mAb 10D5 8 21.68 0.0675 −29 27.29 +/− 19.43 mAb 266 6 16.590.0184 −46 19.59 +/− 6.59 mAb 21F12 8 29.80 >0.9999 −3 32.88 +/− 9.90Footnotes: ^(a). Number of animals per group at the end of theexperiment ^(b). ng/g tissue ^(c). Mann Whitney analysis ^(d). NA: notapplicable ^(e). Standard Deviation

APP concentration was also determined by ELISA in the cortex andcerebellum from antibody-treated and control, PBS-treated mice. Twodifferent APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α, the secreted form of APP which has beencleaved within the Aβ sequence), and full-length forms (FL) of APP,while the second recognizes only APP-α. In contrast to thetreatment-associated diminution of Aβ in a subset of treatment groups,the levels of APP were virtually unchanged in all of the treatedcompared to the control animals. These results indicate that theimmunizations with Aβ antibodies deplete Aβ without depleting APP.

In summary, Aβ levels were significantly reduced in the cortex,hippocampus and cerebellum in animals treated with the polyclonalantibody raised against AN1792. To a lesser extent monoclonal antibodiesto the amino terminal region of Aβ1-42, specifically amino acids 1-16and 13-28 also showed significant treatment effects.

4. Histochemical Analyses

The morphology of Aβ-immunoreactive plaques in subsets of brains frommice in the PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treatment groupswas qualitatively compared to that of previous studies in which standardimmunization procedures with Aβ42 were followed.

The largest alteration in both the extent and appearance of amyloidplaques occurred in the animals immunized with the polyclonal Aβ42antibody. The reduction of amyloid load, eroded plaque morphology andcell-associated Aβ immunoreactivity closely resembled effects producedby the standard immunization procedure. These observations support theELISA results in which significant reductions in both total Aβ and A142were achieved by administration of the polyclonal Aβ42 antibody.

In similar qualitative evaluations, amyloid plaques in the 10D5 groupwere also reduced in number and appearance, with some evidence ofcell-associated Aβ immunoreactivity. Relative to control-treatedanimals, the polyclonal 1 g fraction against Aβ and one of themonoclonal antibodies (10D5) reduced plaque burden by 93% and 81%,respectively (<0.005). 21F12 appeared to have a relatively modest effecton plaque burden. Micrographs of brain after treatment with pabAβ₁₋₄₂show diffuse deposits and absence of many of the larger compactedplaques in the pabAβ₁₋₄₂ treated group relative to control treatedanimals.

5. Measurement of Antibody Titers

A subset of three randomly chosen mice from each group were bled justprior to each intraperitoneal inoculation, for a total of 30 bleeds.Antibody titers were measured as Aβ1-42-binding antibody using asandwich ELISA with plastic multi-well plates coated with Aβ1-42 asdescribed in detail in the General Materials and Methods. Mean titersfor each bleed are shown in FIGS. 16-18 for the polyclonal antibody andthe monoclonals 10D5 and 21 F 12, respectively. Titers averaged about1000 over this time period for the polyclonal antibody preparation andwere slightly above this level for the 10D5- and 21 F 12-treatedanimals.

6. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested eight days following the final antibody infusion. Freshlyharvested cells, 10⁵ per well, were cultured for 5 days in the presenceof Aβ1-40 at a concentration of 5 FM for stimulation. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Splenocytes from aged PDAPP mice passively immunized with variousanti-Aβ antibodies were stimulated in vitro with AN1792 andproliferative and cytokine responses were measured. The purpose of theseassays was to determine if passive immunization facilitated antigenpresentation, and thus priming of T cell responses specific for AN1792.No AN1792-specific proliferative or cytokine responses were observed inmice passively immunized with the anti-Aβ antibodies.

XII: Further Study of Passive Immunization

In a second study, treatment with 10D5 was repeated and two additionalanti-Aβ antibodies were tested, monoclonals 3D6 (Aβ15) and 16C11(Aβ₃₃₋₄₂). Control groups received either PBS or an irrelevantisotype-matched antibody (TM2a). The mice were older (11.5-12 month oldheterozygotes) than in the previous study, otherwise the experimentaldesign was the same. Once again, after six months of treatment, 10D5reduced plaque burden by greater than 80% relative to either the PBS orisotype-matched antibody controls (p=0.003). One of the other antibodiesagainst Aβ, 3D6, was equally effective, producing an 86% reduction(p=0.003). In contrast, the third antibody against the peptide, 16C11,failed to have any effect on plaque burden. Similar findings wereobtained with Aβ₄₂ ELISA measurements. These results demonstrate that anantibody response against Aβ peptide, in the absence of T cell immunity,is sufficient to decrease amyloid deposition in PDAPP mice, but that notall anti-Aβ antibodies are efficacious. Antibodies directed to epitopescomprising amino acids 1-5 or 3-7 of Aβ are particularly efficacious.

In summary, we have shown that passively administered antibodies againstAβ reduced the extent of plaque deposition in a mouse model ofAlzheimer's disease. When held at modest serum concentrations (2570μg/ml), the antibodies gained access to the CNS at levels sufficient todecorate β-amyloid plaques. Antibody entry into the CNS was not due toabnormal leakage of the blood-brain barrier since there was no increasein vascular permeability as measured by Evans Blue in PDAPP mice. Inaddition, the concentration of antibody in the brain parenchyma of agedPDAPP mice was the same as in non-transgenic mice, representing 0.1% ofthe antibody concentration in serum (regardless of isotype).

XIII: Monitoring of Antibody Binding

To determine whether antibodies against AS could be acting directlywithin the CNS, brains taken from saline-perfused mice at the end of theExample XII, were examined for the presence of theperipherally-administered antibodies. Unfixed cryostat brain sectionswere exposed to a fluorescent reagent against mouse immunoglobulin (goatanti-mouse IgG-Cy3). Plaques within brains of the 10D5 and 3D6 groupswere strongly decorated with antibody, while there was no staining inthe 16C11 group. To reveal the full extent of plaque deposition, serialsections of each brain were first immunoreacted with an anti-Aβantibody, and then with the secondary reagent. 10D5 and 3D6, followingperipheral administration, gained access to most plaques within the CNS.The plaque burden was greatly reduced in these treatment groups comparedto the 16C11 group. These data indicate that peripherally administeredantibodies can enter the CNS where they can directly trigger amyloidclearance. It is likely that 16C11 also had access to the plaques butwas unable to bind.

XIV: ex Vivo Screening Assay for Activity of an Antibody Against AmyloidDeposits

To examine the effect of antibodies on plaque clearance, we establishedan ex vivo assay in which primary microglial cells were cultured withunfixed cryostat sections of either PDAPP mouse or human AD brains.Microglial cells were obtained from the cerebral cortices of neonateDBA/2N mice (1-3 days). The cortices were mechanically dissociated inHBSS⁻ (Hanks' Balanced Salt Solution, Sigma) with 50 μg/ml DNase I(Sigma). The dissociated cells were filtered with a 100 μm cell strainer(Falcon), and centrifuged at 1000 rpm for 5 minutes. The pellet wasresuspended in growth medium (high glucose DMEM, 10%FBS, 25ng/mlrmGM-CSF), and the cells were plated at a density of 2 brains per T-75plastic culture flask. After 7-9 days, the flasks were rotated on anorbital shaker at 200 rpm for 2h at 37° C. The cell suspension wascentrifuged at 1000 rpm and resuspended in the assay medium.

10-μm cryostat sections of PDAPP mouse or human AD brains (post-morteminterval<3hr) were thaw mounted onto poly-lysine coated round glasscoverslips and placed in wells of 24-well tissue culture plates. Thecoverslips were washed twice with assay medium consisting of H-SFM(Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5ng/ml rmGM-CSF (R&D). Control or anti-Aβantibodies were added at a 2×concentration (5 μg/ml final) for 1 hour.The microglial cells were then seeded at a density of 0.8×10⁶ cells/mlassay medium. The cultures were maintained in a humidified incubator(37° C., 5%CO₂) for 24hr or more. At the end of the incubation, thecultures were fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton-X100. The sections were stained with biotinylated 3D6 followed bya streptavidin/Cy3 conjugate (Jackson ImmunoResearch). The exogenousmicroglial cells were visualized by a nuclear stain (DAPI). The cultureswere observed with an inverted fluorescent microscope (Nikon, TE300) andphotomicrographs were taken with a SPOT digital camera using SPOTsoftware (Diagnostic instruments). For Western blot analysis, thecultures were extracted in 8M urea, diluted 1:1 in reducing tricinesample buffer and loaded onto a 16% tricine gel (Novex). After transferonto immobilon, blots were exposed to 5 μg/ml of the pabAβ42 followed byan HRP-conjugated anti-mouse antibody, and developed with ECL(Amersham).

When the assay was performed with PDAPP brain sections in the presenceof 16C11 (one of the antibodies against Aβ that was not efficacious invivo), β-amyloid plaques remained intact and no phagocytosis wasobserved. In contrast, when adjacent sections were cultured in thepresence of 10D5, the amyloid deposits were largely gone and themicroglial cells showed numerous phagocytic vesicles containing Aβ.Identical results were obtained with AD brain sections; 10D5 inducedphagocytosis of AD plaques, while 16C11 was ineffective. In addition,the assay provided comparable results when performed with either mouseor human microglial cells, and with mouse, rabbit, or primate antibodiesagainst Aβ.

Table 16 shows whether binding and/or phagocytosis was obtained forseveral different antibody binding specificities. It can be seen thatantibodies binding to epitopes within aa 1-7 both bind and clear amyloiddeposits, whereas antibodies binding to epitopes within amino acids 4-10bind without clearing amyloid deposits. Antibodies binding to epitopesC-terminal to residue 10 neither bind nor clear amyloid deposits.

TABLE 16 Analysis of Epitope Specificity Antibody epitope isotypeStaining Phagocytosis N-Term mab 3D6 1-5 IgG2b + + 10D5 3-6 IgG1 + +22C8 3-7 IgG2a + + 6E10  5-10 IgG1 + − 14A8  4-10 rat IgG1 + − 13-2818G11 10-18 rat IgG1 − − 266 16-24 IgG1 − − 22D12 18-21 IgG2b − − C-Term2G3 −40 IgG1 − − 16C11 −40/−42 IgG1 − − 21F12 −42 IgG2a − − Immune serumrabbit (CFA) 1-6 + + mouse (CFA) 3-7 + + mouse (QS-21) 3-7 + + monkey(QS-21) 1-5 + + mouse (MAP1-7) + +

Table 17 shows results obtained with several antibodies against Aβ,comparing their abilities to induce phagocytosis in the ex vivo assayand to reduce in vivo plaque burden in passive transfer studies.Although 16C11 and 21 F12 bound to aggregated synthetic Aβ peptide withhigh avidity, these antibodies were unable to react with 13-amyloidplaques in unfixed brain sections, could not trigger phagocytosis in theex vivo assay, and were not efficacious in vivo. 10D5, 3D6, and thepolyclonal antibody against Aβ were active by all three measures. The22C8 antibody binds more strongly to an analog form of natural Aβ inwhich aspartic acid at positions 1 and 7 is replaced with iso-asparticacid. These results show that efficacy in vivo is due to direct antibodymediated clearance of the plaques within the CNS, and that the ex vivoassay is predictive of in vivo efficacy.

The same assay has been used to test clearing of an antibody against afragment of synuclein referred to as NAC. Synuclein has been shown to bean amyloid plaque-associated protein. An antibody to NAC was contactedwith a brain tissue sample containing amyloid plaques, an microglialcells, as before. Rabbit serum was used as a control. Subsequentmonitoring showed a marked reduction in the number and size of plaquesindicative of clearing activity of the antibody.

TABLE 17 The ex vivo assay as predictor of in vivo efficacy. Avidity forBinding to aggregated β-amyloid Ex vivo In vivo Antibody Isotype Aβ (pM)plaques efficacy efficacy monoclonal 3D6 IgG2b 470 + + + 10D5 IgG143 + + + 16C11 IgG1 90 − − − 21F12 IgG2a 500 − − − TM2a IgG1 — − − −polyclonal 1-42 mix 600 + + +

Confocal microscopy was used to confirm that Aβ was internalized duringthe course of the ex vivo assay. In the presence of control antibodies,the exogenous microglial cells remained in a confocal plane above thetissue, there were no phagocytic vesicles containing Aβ, and the plaquesremained intact within the section. In the presence of 10D5, nearly allplaque material was contained in vesicles within the exogenousmicroglial cells. To determine the fate of the internalized peptide,10D5 treated cultures were extracted with 8M urea at varioustime-points, and examined by Western blot analysis. At the one hour timepoint, when no phagocytosis had yet occurred, reaction with a polyclonalantibody against Aβ revealed a strong 4 kD band (corresponding to theA13 peptide). Aβ immunoreactivity decreased at day 1 and was absent byday 3. Thus, antibody-mediated. phagocytosis of Aβ leads to itsdegradation.

To determine if phagocytosis in the ex vivo assay was Fc-mediated,F(ab′)2 fragments of the anti-Aβ antibody 3D6 were prepared. Althoughthe F(ab′)2 fragments retained their full ability to react with plaques,they were unable to trigger phagocytosis by microglial cells. Inaddition, phagocytosis with the whole antibody could be blocked by areagent against murine Fc receptors (anti-CD16/32). These data indicatethat in vivo clearance of Aβ occurs through Fc-receptor mediatedphagocytosis.

XV: Passage of Antibodies Through Blood Brain Barrier

This example determines the concentration of antibody delivered to thebrain following intravenous injection into a peripheral tissue of eithernormal or PDAPP mice. PDAPP or control normal mice were perfused with0.9% NaCl. Brain regions (hippocampus or cortex) were dissected andrapidly frozen. Brain were homogenized in 0. 1% triton+proteaseinhibitors. Immunoglobulin was detected in the extracts by ELISA. Fab′2Goat Anti-mouse IgG were coated onto an RIA plate as capture reagent.The serum or the brain extracts were incubated for 1 hr. The isotypeswere detected with anti-mouse IgG1-HRP or IgG2a-HRP or IgG2b-HRP(Caltag). Antibodies, regardless of isotype, were present in the CNS ata concentration that is 1:1000 that found in the blood. For example,when the concentration of IgG1 was three times that of IgG2a in theblood, it was three times IgG2a in the brain as well, both being presentat 0. 1% of their respective levels in the blood. This result wasobserved in both transgenic and nontransgenic mice-so the PDAPP does nothave a uniquely leak blood brain barrier.

XVI: Therapeutic Efficacy of an Aβ Peptide in MAP Configuration

A therapeutic adjuvant/immunogen efficacy study was performed in 9-10.5month old male and female heterozygous PDAPP transgenic mice to test theefficacy of a fusion protein comprising Aβ1-7 in tetrameric MAPconfiguration as described above. The duration of the study was 25 weekswith 29-40 animals per treatment group; therefore the animals were15-16.5 months old at termination. The methodology used in this study isthe same as that in the therapeutic study of different adjuvants inExample VIII above. The treatment groups are identified in Table 18below.

TABLE 18 Dilution Adjuvant Immunogen Buffer Administration Group 1:CFA/IFA MAP(Aβ 1-7:TT) PBS IP (400 μl) (100 μg) Group 2: QS21 AN1792-GCS(75 μg) PBS SC (250 μl) Group 3: PBS — — SC (250 μl) Tableabbreviations: MAP - multi-antigenic peptide; TT - tetanus toxoid t-cellepitope (830-844); SC - subcutaneous; IP - intraperitoneally; PBS -phosphate buffered saline; GCS is a glycine/citrate/sucrose formulation.

The immunization schedule was identical for all of the treatment groups.The mice were injected on weeks 0, 2, 4, 8, 12, 16, 20, 24, with bleedson week 3, 5, 9, 13, 17, 21 and 25. Groups 1, 2, 3, 4, and 6 receivedeight injections Groups 2 and 3, QS21/AN1792 and PBS respectively,served as the positive and negative controls for this study.

The titers were determined by the anti-Aβ antibody titer assay.

Group 1, CFA/IFA:MAP(Aβ1-7:TT) group, had low titer levels. The peak GMTreached was only 1,200 at 13 weeks, falling to a GMT of 600 by week 25.There were 3 of the 30 mice that did not raise any titer and another 7mice that did not exceed a titer of 400 by the end of the study.

Group 2, the QS21/AN1792 control group, reached its peak titer at 17weeks with a GMT of 16,000. The titer then fell over the next 8 weeks tofinish with a GMT of 8,700. One animal in this group failed to raise atiter over the entire course of the experiment.

Group 3, the PBS alone group, had no titers.

Both treatment groups showed a significant lowing in cortical Aβ levelsas compared to the PBS control group (see Table 19). TheCFA/IFA:MAP(Aβ1-7) group, significantly lowered Aβ as compared to thePBS control group in spite of the relatively low titers of anti-Aβantibodies.

TABLE 19 Cortical Aβ levels PBS MAP QS-21 MEDIAN 7,335 3,692 2,389 (ng/gtissue) RANGE 550-18,358 240-10,782 210-11,167 (ng/g tissue) p value —0.0003 <0.0001 N 38 30 34

In conclusion, the Aβ1-7MAP immunogen is effective in inducing asufficient immune response significantly to retard Aβ deposition in thecortex.

XVII. Epitope Mapping of Immunogenic Response to Aβ in Monkeys

This example analyzes the response of a primate to immunization withAN1792 (i.e., Aβ1-42). Eleven groups of monkeys (4/sex/group) wereimmunized with AN1792 (75 or 300 μg/dose) in combination with QS-21adjuvant (50 or 100 μg/dose) or 5% sterile dextrose in water (D5W,control group). All animals received IM injections on one of threeinjection schedules as shown in Table 20 for a total of 4, 5 or 8 doses.Serum samples (from 4 monkeys/sex/group) collected on Day 175 of thestudy and CSF samples (from 3 monkeys/sex/group) collected on Day 176 ofthe study (at the 6 month necropsy) were evaluated for their ability tobind to Aβ1-40 peptide and APP.

TABLE 20 Group Assignments and Dose Levels Group # Monkeys AN1792 DoseQS-21 Dose Dose No. Schedule^(a) (M/F) (μg/dose) (μg/dose) Route  1^(b)1 4/4 0 0 IM 2 1 4/4 Vehicle^(c) 50 IM 3 1 4/4 Vehicle 100 IM 4 1 4/4 7550 IM 5 1 4/4 300 50 IM 6 1 4/4 75 100 IM 7 1 4/4 300 100 IM 8 2 4/4 75100 IM 9 2 4/4 300 100 IM 10  3 4/4 75 100 IM 11  3 4/4 300 100 IM ^(a).Schedule 1, Dose Days 1, 15, 29, 57, 85, 113, 141, 169; Schedule 2, DoseDays 1, 29, 57, 113, 169; Schedule 3, Dose Days 1, 43, 85, 169 ^(b). D5Winjection control group ^(c). Vehicle consists of theglycine/citrate/sucrose buffer which is the excipient for AN1792.

The exact array of linear peptides recognized by the antibodies in theserum samples from animals immunized with AN1792 was determined by anELISA that measured the binding of these antibodies to overlappingpeptides that covered the entire Aβ1-42 sequence. Biotinylated peptideswith partial sequences of AN1792 were obtained from Chiron Technologiesas 10 amino acid peptides with an overlap of 9 residues and a step ofone residue per peptide (synthesis No. 5366, No. 5331 and No. 5814). Thefirst 32 peptides (from the eight amino acid position upstream of theN-terminal of AN1792 down to the twenty-fourth amino acid of AN1792) arebiotinylated on the C-terminal with a linker of GGK. The last 10peptides (repeating the thirty-second peptide from the previous series)are biotinylated on the N-terminal with a linker consisting of EGEG (SEQID NO:76). The lyophilized biotinylated peptides were dissolved at aconcentration of 5 mM in DMSO. These peptide stocks were diluted to 5 μMin TTBS (0.05% Tween 20,25 mM Tris HCl, 137 mM NaCl, 5.1 mM KCl,pH=7.5). 100 μl aliquots of this 5 μM solution were added in duplicateto streptavidin pre-coated 96-well plates (Pierce). Plates wereincubated for one hour at room temperature, then washed four times withTTBS. Serum samples were diluted in specimen diluent without azide tonormalize titers, and 100 μl was added per well. These plates wereincubated one hour at room temperature and then washed four times withTTBS. HRP-conjugated goat anti-human antibody (Jackson ImmunoResearch)was diluted 1:10,000 in specimen diluent without azide and 100 μl wasadded per well. The plates were again incubated and washed. To developthe color reaction, TMB (Pierce), was added at 100 μl per well andincubated for 15 min prior to the addition of 30 μl of 2 N H₂SO₄ to stopthe reaction.

The optical density was measured at 450 nm on a Vmax or Spectramaxcolorimetric plate reader.

Immunization with AN1792 resulted in the production of antibodies in100% of the animals in all of the dose groups by Day 175. Mean titers inthe groups ranged from 14596-56084. There was a trend for titers to behigher within an immunization schedule in the presence of higher antigenand/or higher adjuvant concentration, but no statistically significantdifferences could be demonstrated due to the high variability inindividual animal responses to the immunizations.

Sera which were positive for antibodies to AN1792 were also positive forantibodies to Aβ1-40. Mean titers in the groups ranged from36867-165991, and as for anti-AN1792 titers, showed no statisticallysignificant differences between groups at Day 175. Binding to AN1792showed a highly positive correlation (Spearman r=0.8671) with binding toAβ1-40.

Of the 48 monkeys immunized on various schedules with AN1792, 33 yieldedCSF samples of adequate volume and quality for analysis. Thirty-two(97%) of these monkeys had positive titers to AN1792. Titers ranged from2-246, with a mean of 49.44±21.34. CSF anti-AN1792 levels were0.18±0.11% of what was measured in the serum and demonstrated a highlypositive correlation (Spearman r=0.7840) with serum titers. Nodifferences were seen across groups or between sexes in the percentageof antibody in the CSF. The level of antibody in the CSF is consistentwith the passive transfer of peripherally generated antibody across theblood-brain-barrier into the central nervous system.

Testing of a subset of anti-AN1792 positive CSF samples demonstratedthat, like the antibody in serum samples, antibody in the CSFcross-reacts with Aβ1-40. Titers to Aβ1-40 showed a high correlation(Spearman r=0.9634) to their respective AN1792 titers. Testing of asubset of CSF samples with the highest titers to AN1792 showed nobinding to APP, as for the serum antibodies.

When sera from Day 175 was tested against a series of overlapping 10-merpeptides, antibodies from all of the monkeys bound to the peptide whosesequence covered amino acids 1-10 of the AN1792 peptide (amino acids653-672 of APP). In some animals, this was the only peptide to whichbinding could be measured (see FIG. 19).

In other animals, other reactivities could be measured, but in all casesthe reactivity to the N-terminal peptide sequence was the predominantone. The additional reactivities fell into two groups. First and mostcommon, was the binding to peptides centering around the N-terminal 1-10AN1792 peptide (FIG. 20). Binding of this type was directed to thepeptides covering amino acids -1-8, -1-9, and 2-11 of the AN1792peptide. These reactivities, combined with that to the 1-10 peptide,represent the overwhelming majority of reactivity in all animals.Epitope mapping of individual animals over time indicates that theantibody reactivity to the 1-10 peptide proceeds the spread to theadjacent peptides. This demonstrates a strong biasing of the immuneresponse to the N-terminus of the AN1792 peptide with its free terminalaspartic acid residue. The second minor detectable activity in someanimals was binding to peptides located C-terminally to the major areaand centered around peptides covering amino acids 7-16, 11-20 and 16-25of the AN1792 peptide . These reactivities were seen in only 10-30% ofthe monkeys.

Variability in response between different animals (e.g., whether aminoacids 1-10 were the exclusive or predominant reactive epitope) did notcorrelate with antigen/adjuvant dose, dosing schedule, or antibodytiter, and is probably a reflection of each individual animal's geneticmake-up.

XVIII. Prevention and Treatment of Human Subjects

A single-dose phase I trial is performed to determine safety in humans.A therapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAβ are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measures, patients begin receiving treatment. Theyare randomized and treated with either therapeutic agent or placebo in ablinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase 11 trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI) or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

XIX. General Materials and Methods

1. Measurement of Antibody Titers

Mice were bled by making a small nick in the tail vein and collectingabout 200 μl of blood into a microfuge tube. Guinea pigs were bled byfirst shaving the back hock area and then using an 18 gauge needle tonick the metatarsal vein and collecting the blood into microfuge tubes.Blood was allowed to clot for one hr at room temperature (RT), vortexed,then centrifuged at 14,000 ×g for 10 min to separate the clot from theserum. Serum was then transferred to a clean microfuge tube and storedat 4° C. until titered.

Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 7.4,0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serialdilutions of the samples were made directly in the plates in three-foldsteps to reach a final dilution of 1/218,700. The dilutions wereincubated in the coated-plate wells for one hr at RT. The plates werethen washed four times with PBS containing 0.05% Tween 20. The secondantibody, a goat anti-mouse Ig conjugated to horseradish peroxidase(obtained from Boehringer Mannheim), was added to the wells as 100 μl ofa 1/3000 dilution in Specimen Diluent and incubated for one hr at RT.Plates were again washed four times in PBS, Tween 20. To develop thechromogen, 100 μl of Slow TMB (3,3′,5,5′-tetramethyl benzidine obtainedfrom Pierce Chemicals) was added to each well and incubated for 15 minat RT. The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄.The color intensity was then read on a Molecular Devices Vmax at (450nm-650 nm).

Titers were defined as the reciprocal of the dilution of serum givingone half the maximum OD. Maximal OD was generally taken from an initial1/100 dilution, except in cases with very high titers, in which case ahigher initial dilution was necessary to establish the maximal OD. Ifthe 50% point fell between two dilutions, a linear extrapolation wasmade to calculate the final titer. To calculate geometric mean antibodytiters, titers less than 100 were arbitrarily assigned a titer value of25.

2. Lymphocyte proliferation assay

Mice were anesthetized with isoflurane. Spleens were removed and rinsedtwice with 5 ml PBS containing 10% heat-inactivated fetal bovine serum(PBS-FBS) and then homogenized in a 50° Centricon unit (Dako A/S,Denmark) in 1.5 ml PBS-FBS for 10 sec at 100 rpm in a Medimachine (Dako)followed by filtration through a 100 micron pore size nylon mesh.Splenocytes were washed once with 15 ml PBS-FBS, then pelleted bycentrifugation at 200 ×g for 5 min. Red blood cells were lysed byresuspending the pellet in 5 mL buffer containing 0.15 M NH4C1, 1 MKHCO3, 0.1 M NaEDTA, pH 7.4 for five min at RT. Leukocytes were thenwashed as above. Freshly isolated spleen cells (105 cells per well) werecultured in triplicate sets in 96-well U-bottomed tissue culture-treatedmicrotiter plates (Coming, Cambridge, Mass.) in RPMI1640 medium (JRHBiosciences, Lenexa, Kans.) supplemented with 2.05 mM L glutamine, 1%Penicillin/Streptomycin, and 10% heat-inactivated FBS, for 96 hr at 37°C. Various Aβ peptides, Aβ1-16, Aβ1-40, Aβ1-42 or Aβ40-1 reversesequence protein were also added at doses ranging from 5 to 0.18micromolar in four steps. Cells in control wells were cultured withConcanavalin A (Con A) (Sigma, cat. # C-5275, at 1 microgram/ml) withoutadded protein. Cells were pulsed for the final 24 hr with 3H-thymidine(1 μCi/well obtained from Amersham Corp., Arlington Heights Ill.). Cellswere then harvested onto UniFilter plates and counted in a Top CountMicroplate Scintillation Counter (Packard Instruments, Downers Grove,Ill.). Results are expressed as counts per minute (cpm) of radioactivityincorporated into insoluble macromolecules.

4. Brain Tissue Preparation

After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

Tissues destined for ELISAs were homogenized in 10 volumes of ice-coldguanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Thehomogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to four hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse litterrnates(Johnson-Wood et al., supra).

5. Measurement of Aβ Levels

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000 ×g for 20 min at4° C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody (mAb) 266, specific for amino acids 13-28of Aβ (Seubert, et al.), as the capture antibody, and biotinylated mAb3D6, specific for amino acids 1-5 of Aβ (Johnson-Wood, et al.), as thereporter antibody. The 3D6 mAb does not recognize secreted APP orfull-length APP, but detects only Aβ species with an amino-terminalaspartic acid. The cell line producing the antibody 3D6 has the ATCCaccession number PTA-5130, having been deposited on Apr. 8, 2003. Thisassay has a lower limit of sensitivity of 50 ng/ml (II nM) and shows nocross-reactivity to the endogenous murine Aβ protein at concentrationsup to 1 ng/ml (Johnson-Wood et al., supra).

The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specific for aminoacids 33-42 of Aβ (Johnson-Wood, et al.), as the capture antibody.Biotinylated mAβ 3D6 is also the reporter antibody in this assay whichhas a lower limit of sensitivity of about 125 μg/ml (28 μM, Johnson-Woodet al.). For the Aβ ELISAs, 100 μl of either mAβ 266 (at 10 μg/ml) ormAβ 21F12 at (5 μg/ml) was coated into the wells of 96-well immunoassayplates (Costar) by overnight incubation at RT. The solution was removedby aspiration and the wells were blocked by the addition of 200 μl of0.25% human serum albumin in PBS buffer for at least 1 hr at RT.Blocking solution was removed and the plates were stored desiccated at4° C. until used. The plates were rehydrated with Wash Buffer[Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 0.05%Tween 20] prior to use. The samples and standards were added intriplicate aliquots of 100 μl per well and then incubated overnight at4° C. The plates were washed at least three times with Wash Bufferbetween each step of the assay. The biotinylated mAβ3D6, diluted to 0.5μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The colorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 N H2SO4. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

6. Measurement of APP Levels

Two different APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α) and full-length (FL) forms of APP. Thesecond assay is specific for APP-α. The APP-α/FL assay recognizessecreted APP including the first 12 amino acids of Aβ. Since thereporter antibody (2H3) is not specific to the α-clip-site, occurringbetween amino acids 612-613 of APP695 (Esch et al., Science 248,1122-1124 (1990)); this assay also recognizes full length APP (APP-FL).Preliminary experiments using immobilized APP antibodies to thecytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAb 8E5, raised against amino acids 444 to 592 of the APP695 form (Gameset al., supra). The reporter mAb for the APP-α/FL assay is mAb 2H3,specific for amino acids 597-608 of APP695 (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAb 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-αFL assay is about 11 ng/ml (150ρM) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAb 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAb 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor tm 2350 fluorimeter (Millipore)at 365 nm excitation and 450 nm emission.

7. Immunohistochemistry

Brains were fixed for three days at 40C in 4% paraformaldehyde in PBSand then stored from one to seven days at 4° C. in 1% paraformaldehyde,PBS until sectioned. Forty-micron-thick coronal sections were cut on avibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethyleneglycol in phosphate buffer) at −20° C. prior to immunohistochemicalprocessing. For each brain, six sections at the level of the dorsalhippocampus, each separated by consecutive 240 μm intervals, wereincubated overnight with one of the following antibodies: (1) abiotinylated anti-Aβ (mAb, 3D6, specific for human Aβ) diluted to aconcentration of 2 μg/ml in PBS and 1% horse serum; or (2) abiotinylated mAb specific for human APP, 8E5, diluted to a concentrationof 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAb specific for glialfibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4(TBS); or (4) a mAb specific for CD 11 b, MAC-1 antigen, (ChemiconInternational) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serumin TBS; or (5) a mAb specific for MHC II antigen, (Pharmingen) diluted1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a ratmAb specific for CD 43 (Pharmingen) diluted 1:100 with 1% rabbit serumin PBS or (7) a rat Aβ specific for CD 45RA (Pharmingen) diluted 1:100with 1% rabbit serum in PBS; or (8) a rat monoclonal Aβ specific for CD45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) arat monoclonal Aβ specific for CD 45 (Pharmingen) diluted 1:100 with 1%rabbit serum in PBS; or (10) a biotinylated polyclonal hamster Aβspecific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBSor (11) a rat mAb specific for CD3 (Serotec) diluted 1:200 with 1%rabbit serum in PBS; or with (12) a solution of PBS lacking a primaryantibody containing 1% normal horse serum.

Sections reacted with antibody solutions listed in 1,2 and 6-12 abovewere pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBSfor 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAbs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific for CD45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingamne, Calif.).

Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1- AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1-or MHC II-specific monoclonal antibody as the primaryantibody were subsequently reacted for 1 hr at RT with biotinylatedanti-rat IgG made in rabbit diluted 1:200 with TBS, followed byincubation for one hr with avidin-biotin-peroxidase complex diluted1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHCII-specific antibodies were then visualized by treatment at RT with0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and11 min, respectively.

Immunolabeled sections were mounted on glass slides (VWR, Superfrostslides), air dried overnight, dipped in Propar (Anatech) and overlaidwith coverslips using Permount (Fisher) as the mounting medium.

To counterstain Aβ plaques, a subset of the GFAP-positive sections weremounted on Superfrost slides and incubated in aqueous 1% Thioflavin S(Sigma) for 7 min following immunohistochemical processing. Sectionswere then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

8. Image Analysis

A Videometric 150 Image Analysis System (Oncor, Inc., Gaithersburg, Md.)linked to a Nikon Microphot-FX microscope through a CCD video camera anda Sony Trinitron monitor was used for quantification of theimmunoreactive slides. The image of the section was stored in a videobuffer and a color-and saturation-based threshold was determined toselect and calculate the total pixel area occupied by the immunolabeledstructures. For each section, the hippocampus was manually outlined andthe total pixel area occupied by the hippocampus was calculated. Thepercent amyloid burden was measured as: (the fraction of the hippocampalarea containing Aβ deposits immunoreactive with mAb 3D6)×100. Similarly,the percent neuritic burden was measured as: (the fraction of thehippocampal area containing dystrophic neurites reactive with monoclonalantibody 8E5) ×100. The C-Imaging System (Compix, Inc., CranberryTownship, Pa.) operating the Simple 32 Software Application program waslinked to a Nikon Microphot-FX microscope through an Optronics cameraand used to quantitate the percentage of the retrospenial cortexoccupied by GFAP-positive astrocytes and MAC-1-and MHC II-positivemicroglia. The image of the immunoreacted section was stored in a videobuffer and a monochrome-based threshold was determined to select andcalculate the total pixel area occupied by immunolabeled cells. For eachsection, the retrosplenial cortex (RSC) was manually outlined and thetotal pixel area occupied by the RSC was calculated. The percentastrocytosis was defined as: (the fraction of RSC occupied byGFAP-reactive astrocytes) X 100. Similarly, percent microgliosis wasdefined as: (the fraction of the RSC occupied by MAC-1- or MHCII-reactive microglia) X 100. For all image analyses, six sections atthe level of the dorsal hippocampus, each separated by consecutive 240μm intervals, were quantitated for each animal. In all cases, thetreatment status of the animals was unknown to the observer.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

From the foregoing it will be apparent that the invention provides for anumber of uses. For example, the invention provides for the use of anyof the antibodies to Aβ described above in the treatment, prophylaxis ordiagnosis of amyloidogenic disease, or in the manufacture of amedicament or diagnostic composition for use in the same. Likewise, theinvention provides for the use of any of the epitopic fragments of Aβdescribed above for the treatment or prophylaxis of amyloidogenicdisease or in the manufacture of a medicament for use in the same.

TABLE 1 TITER AT 50% MAXIMAL O.D. Aggreated Aβ injected mice Age ofPDAPP mouse 100 mouse 101 mouse 102 mouse 103 mouse 104 mouse 105 mouse106 mouse 107 mouse 108 4 70000 150000 15000 120000 1000 15000 5000080000 100000 6 15000 65000 30000 55000 300 15000 15000 50000 60000 820000 55000 50000 50000 400 15000 18000 50000 60000 10 40000 20000 6000050000 900 15000 50000 20000 40000 12 25000 30000 60000 40000 2700 2000070000 25000 20000 PBS injected mice on both immunogens; at 1/100 Age ofPDAPP mouse 113 mouse 114 mouse 115 mouse 116 mouse 117 6 <4x bkg <4xbkg <4x bkg <4x bkg <4x bkg 10   5x bkg <4x bkg <4x bkg <4x bkg <4x bkg12 <4x bkg <4x bkg <4x bkg <4x bkg <4x bkg

77 1 10 PRT Artificial Sequence Description of Artificial Sequence10-merpeptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 1 GluGlu Ile Ser Glu Val Lys Met Asp Ala 1 5 10 2 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 2 Glu Ile Ser Glu Val Lys Met AspAla Glu 1 5 10 3 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 3 Ile Ser Glu Val Lys Met Asp Ala Glu Phe 1 5 10 4 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 4 Ser Glu ValLys Met Asp Ala Glu Phe Arg 1 5 10 5 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 5 Glu Val Lys Met Asp Ala Glu PheArg His 1 5 10 6 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 6 Val Lys Met Asp Ala Glu Phe Arg His Asp 1 5 10 7 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 7 Lys Met AspAla Glu Phe Arg His Asp Ser 1 5 10 8 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 8 Met Asp Ala Glu Phe Arg His AspSer Gly 1 5 10 9 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 9 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 1 5 10 10 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 10 Ala GluPhe Arg His Asp Ser Gly Tyr Glu 1 5 10 11 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 11 Glu Phe Arg His Asp Ser Gly TyrGlu Val 1 5 10 12 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 12 Phe Arg His Asp Ser Gly Tyr Glu Val His 1 5 10 13 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 13 Arg HisAsp Ser Gly Tyr Glu Val His His 1 5 10 14 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 14 His Asp Ser Gly Tyr Glu Val HisHis Gln 1 5 10 15 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 15 Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 16 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 16 Ser GlyTyr Glu Val His His Gln Lys Leu 1 5 10 17 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 17 Gly Tyr Glu Val His His Gln LysLeu Val 1 5 10 18 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 18 Tyr Glu Val His His Gln Lys Leu Val Phe 1 5 10 19 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 19 Glu ValHis His Gln Lys Leu Val Phe Phe 1 5 10 20 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 20 Val His His Gln Lys Leu Val PhePhe Ala 1 5 10 21 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 21 His His Gln Lys Leu Val Phe Phe Ala Glu 1 5 10 22 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 22 His GlnLys Leu Val Phe Phe Ala Glu Asp 1 5 10 23 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 23 Gln Lys Leu Val Phe Phe Ala GluAsp Val 1 5 10 24 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 24 Lys Leu Val Phe Phe Ala Glu Asp Val Gly 1 5 10 25 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 25 Leu ValPhe Phe Ala Glu Asp Val Gly Ser 1 5 10 26 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 26 Val Phe Phe Ala Glu Asp Val GlySer Asn 1 5 10 27 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 27 Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 1 5 10 28 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 28 Phe AlaGlu Asp Val Gly Ser Asn Lys Gly 1 5 10 29 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 29 Ala Glu Asp Val Gly Ser Asn LysGly Ala 1 5 10 30 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 30 Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 1 5 10 31 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 31 Asp ValGly Ser Asn Lys Gly Ala Ile Ile 1 5 10 32 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 32 Val Gly Ser Asn Lys Gly Ala IleIle Gly 1 5 10 33 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 33 Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu 1 5 10 34 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 34 Ser AsnLys Gly Ala Ile Ile Gly Leu Met 1 5 10 35 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 35 Asn Lys Gly Ala Ile Ile Gly LeuMet Val 1 5 10 36 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 36 Lys Gly Ala Ile Ile Gly Leu Met Val Gly 1 5 10 37 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 37 Gly AlaIle Ile Gly Leu Met Val Gly Gly 1 5 10 38 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 38 Ala Ile Ile Gly Leu Met Val GlyGly Val 1 5 10 39 10 PRT Artificial Sequence Description of ArtificialSequence10-mer peptide from AN1792 sequence (human Abeta42, beta-amyloidpeptide) 39 Ile Ile Gly Leu Met Val Gly Gly Val Val 1 5 10 40 10 PRTArtificial Sequence Description of Artificial Sequence10-mer peptidefrom AN1792 sequence (human Abeta42, beta-amyloid peptide) 40 Ile GlyLeu Met Val Gly Gly Val Val Ile 1 5 10 41 10 PRT Artificial SequenceDescription of Artificial Sequence10-mer peptide from AN1792 sequence(human Abeta42, beta-amyloid peptide) 41 Gly Leu Met Val Gly Gly Val ValIle Ala 1 5 10 42 42 PRT Homo sapiens human Abeta42 beta-amyloid peptide42 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 510 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 2025 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 43 13 PRT ArtificialSequence Description of Artificial Sequenceinfluenza hemagglutininHA-307-319 universal T-cell epitope 43 Pro Lys Tyr Val Lys Gln Asn ThrLeu Lys Leu Ala Thr 1 5 10 44 13 PRT Artificial Sequence Description ofArtificial SequencePADRE universal T-cell epitope 44 Ala Lys Xaa Val AlaAla Trp Thr Leu Lys Ala Ala Ala 1 5 10 45 16 PRT Artificial SequenceDescription of Artificial Sequencemalaria CS, T3 epitope universalT-cell epitope 45 Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser ValPhe Asn Val 1 5 10 15 46 10 PRT Artificial Sequence Description ofArtificial Sequencehepatitis B surface antigen HBsAg-19-28 universalT-cell epitope 46 Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile 1 5 10 47 19PRT Artificial Sequence Description of Artificial Sequenceheat shockprotein 65 hsp65-153-171 universal T-cell epitope 47 Asp Gln Ser Ile GlyAsp Leu Ile Ala Glu Ala Met Asp Lys Val Gly 1 5 10 15 Asn Glu Gly 48 14PRT Artificial Sequence Description of Artificial SequencebacilleCalmette-Guerin universal T-cell epitope 48 Gln Val His Phe Gln Pro LeuPro Pro Ala Val Val Lys Leu 1 5 10 49 15 PRT Artificial SequenceDescription of Artificial Sequencetetanus toxoid TT-830-844 universalT-cell epitope 49 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile ThrGlu Leu 1 5 10 15 50 21 PRT Artificial Sequence Description ofArtificial Sequencetetanus toxoid TT-947-967 universal T-cell epitope 50Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5 1015 Ala Ser His Leu Glu 20 51 16 PRT Artificial Sequence Description ofArtificial SequenceHIV gp120 T1 universal T-cell epitope 51 Lys Gln IleIle Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15 52 22 PRTArtificial Sequence Description of Artificial SequenceAN 90549 Abeta1-7/tetanus toxoid 830-844 52 Asp Ala Glu Phe Arg His Asp Gln Tyr IleLys Ala Asn Ser Lys Phe 1 5 10 15 Ile Gly Ile Thr Glu Leu 20 53 28 PRTArtificial Sequence Description of Artificial SequenceAN 90550 Abeta1-7/tetanus toxoid 947-967 53 Asp Ala Glu Phe Arg His Asp Phe Asn AsnPhe Thr Val Ser Phe Trp 1 5 10 15 Leu Arg Val Pro Lys Val Ser Ala SerHis Leu Glu 20 25 54 43 PRT Artificial Sequence Description ofArtificial SequenceAN90542 Abeta 1-7/tetanus toxoid 830-844 + 947-967 54Asp Ala Glu Phe Arg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe 1 5 1015 Ile Gly Ile Thr Glu Leu Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 20 2530 Arg Val Pro Lys Val Ser Ala Ser His Leu Glu 35 40 55 22 PRTArtificial Sequence Description of Artificial SequenceAN 90576 Abeta3-9/tetanus toxoid 830-844 55 Glu Phe Arg His Asp Ser Gly Gln Tyr IleLys Ala Asn Ser Lys Phe 1 5 10 15 Ile Gly Ile Thr Glu Leu 20 56 20 PRTArtificial Sequence Description of Artificial SequenceAN90562 Abeta1-7/peptide 56 Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala AspAla Glu 1 5 10 15 Phe Arg His Asp 20 57 34 PRT Artificial SequenceDescription of Artificial SequenceAN90543 Abeta 1-7 x 3/peptide 57 AspAla Glu Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala 1 5 10 15Glu Phe Arg His Asp Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala 20 25 30Ala Ala 58 34 PRT Artificial Sequence Description of ArtificialSequencefusion protein with Abeta epitope 58 Ala Lys Xaa Val Ala Ala TrpThr Leu Lys Ala Ala Ala Asp Ala Glu 1 5 10 15 Phe Arg His Asp Asp AlaGlu Phe Arg His Asp Asp Ala Glu Phe Arg 20 25 30 His Asp 59 20 PRTArtificial Sequence Description of Artificial Sequencefusion proteinwith Abeta epitope 59 Asp Ala Glu Phe Arg His Asp Ala Lys Xaa Val AlaAla Trp Thr Leu 1 5 10 15 Lys Ala Ala Ala 20 60 24 PRT ArtificialSequence Description of Artificial Sequencefusion protein with Abetaepitope 60 Asp Ala Glu Phe Arg His Asp Ile Ser Gln Ala Val His Ala AlaHis 1 5 10 15 Ala Glu Ile Asn Glu Ala Gly Arg 20 61 24 PRT ArtificialSequence Description of Artificial Sequencefusion protein with Abetaepitope 61 Phe Arg His Asp Ser Gly Tyr Ile Ser Gln Ala Val His Ala AlaHis 1 5 10 15 Ala Glu Ile Asn Glu Ala Gly Arg 20 62 24 PRT ArtificialSequence Description of Artificial Sequencefusion protein with Abetaepitope 62 Glu Phe Arg His Asp Ser Gly Ile Ser Gln Ala Val His Ala AlaHis 1 5 10 15 Ala Glu Ile Asn Glu Ala Gly Arg 20 63 34 PRT ArtificialSequence Description of Artificial Sequencefusion protein with Abetaepitope 63 Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Asp AlaGlu 1 5 10 15 Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala GluPhe Arg 20 25 30 His Asp 64 27 PRT Artificial Sequence Description ofArtificial Sequencefusion protein with Abeta epitope 64 Asp Ala Glu PheArg His Asp Pro Lys Tyr Val Lys Gln Asn Thr Leu 1 5 10 15 Lys Leu AlaThr Asp Ala Glu Phe Arg His Asp 20 25 65 34 PRT Artificial SequenceDescription of Artificial Sequencefusion protein with Abeta epitope 65Asp Ala Glu Phe Arg His Asp Asp Ala Glu Phe Arg His Asp Asp Ala 1 5 1015 Glu Phe Arg His Asp Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu 20 2530 Ala Thr 66 27 PRT Artificial Sequence Description of ArtificialSequencefusion protein with Abeta epitope 66 Asp Ala Glu Phe Arg His AspAsp Ala Glu Phe Arg His Asp Pro Lys 1 5 10 15 Tyr Val Lys Gln Asn ThrLeu Lys Leu Ala Thr 20 25 67 79 PRT Artificial Sequence Description ofArtificial Sequencefusion protein with Abeta epitope 67 Asp Ala Glu PheArg His Asp Pro Lys Tyr Val Lys Gln Asn Thr Leu 1 5 10 15 Lys Leu AlaThr Glu Lys Lys Ile Ala Lys Met Glu Lys Ala Ser Ser 20 25 30 Val Phe AsnVal Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 35 40 45 Thr Glu LeuPhe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro 50 55 60 Lys Val SerAla Ser His Leu Glu Asp Ala Glu Phe Arg His Asp 65 70 75 68 57 PRTArtificial Sequence Description of Artificial Sequencefusion proteinwith Abeta epitope 68 Asp Ala Glu Phe Arg His Asp Asp Ala Glu Phe ArgHis Asp Asp Ala 1 5 10 15 Glu Phe Arg His Asp Gln Tyr Ile Lys Ala AsnSer Lys Phe Ile Gly 20 25 30 Ile Thr Glu Leu Phe Asn Asn Phe Thr Val SerPhe Trp Leu Arg Val 35 40 45 Pro Lys Val Ser Ala Ser His Leu Glu 50 5569 44 PRT Artificial Sequence Description of Artificial Sequencefusionprotein with Abeta epitope 69 Asp Ala Glu Phe Arg His Asp Gln Tyr IleLys Ala Asn Ser Lys Phe 1 5 10 15 Ile Gly Ile Thr Glu Leu Cys Phe AsnAsn Phe Thr Val Ser Phe Trp 20 25 30 Leu Arg Val Pro Lys Val Ser Ala SerHis Leu Glu 35 40 70 51 PRT Artificial Sequence Description ofArtificial Sequencefusion protein with Abeta epitope 70 Asp Ala Glu PheArg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe 1 5 10 15 Ile Gly IleThr Glu Leu Cys Phe Asn Asn Phe Thr Val Ser Phe Trp 20 25 30 Leu Arg ValPro Lys Val Ser Ala Ser His Leu Glu Asp Ala Glu Phe 35 40 45 Arg His Asp50 71 26 PRT Artificial Sequence Description of ArtificialSequencesynuclein fusion protein 71 Glu Gln Val Thr Asn Val Gly Gly AlaIle Ser Gln Ala Val His Ala 1 5 10 15 Ala His Ala Glu Ile Asn Glu AlaGly Arg 20 25 72 13 PRT Artificial Sequence Description of ArtificialSequenceAbeta 1-12 peptide with inserted Cys residue 72 Asp Ala Glu PheArg His Asp Ser Gly Tyr Glu Val Cys 1 5 10 73 6 PRT Artificial SequenceDescription of Artificial SequenceAbeta 1-5 peptide with inserted Cysresidue 73 Asp Ala Glu Phe Arg Cys 1 5 74 12 PRT Artificial SequenceDescription of Artificial SequenceAbeta 33-42 peptide with inserted Cysresidue 74 Cys Xaa Gly Leu Met Val Gly Gly Val Val Ile Ala 1 5 10 75 19PRT Artificial Sequence Description of Artificial SequenceAbeta 13-28peptide with two Gly residues added and inserted Cys residue 75 Xaa HisGln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 1 5 10 15 GlyGly Cys 76 4 PRT Artificial Sequence Description of ArtificialSequencelinker 76 Glu Gly Glu Gly 1 77 22 PRT Artificial SequenceDescription of Artificial Sequencefusion protein with Abeta epitope 77Asp Ala Glu Phe Arg His Asp Gln Tyr Ile Lys Ala Asn Ser Lys Phe 1 5 1015 Ile Gly Ile Thr Glu Leu 20

What is claimed is:
 1. A method of prophylactically or therapeuticallytreating Alzheimer's disease, comprising administering to the patient aneffective dosage of a pharmaceutical composition comprising a humanizedor chimeric antibody that specifically binds to an epitope within Aβ1-7,and hereby prophylactically or therapeutically treating the patient. 2.The method of claim 1, wherein the antibody is of human isotype IgG1. 3.The method of any one of the preceding claims, wherein the patient ishuman.
 4. The method of claim 1, wherein the antibody specifically bindsto an epitope within residues 1-6 of Aβ.
 5. The method of claim 1,wherein the antibody specifically binds to an epitope within residues1-5 of Aβ.
 6. The method of claim 1, wherein the antibody specificallybinds to an epitope within residues 3-7 of Aβ.
 7. The method of claim 1,wherein the antibody specifically binds to an epitope within residues1-3 of Aβ.
 8. The method of claim 1, wherein the antibody specificallybinds to an epitope within residues 14 of Aβ.
 9. The method of claim 1,wherein the patient is asymptomatic.
 10. The method of claim 1, whereinthe patient is under
 50. 11. The method of claim 1, wherein the patienthas inherited risk factors indicating susceptibility to Alzheimer'sdisease.
 12. The method of claim 1, wherein the patient has no knownrisk factors for Alzheimer's disease.
 13. The method of claim 1, whereinthe antibody is a human antibody.
 14. The method of claim 1, wherein theantibody is a humanized antibody.
 15. The method of claim 1, wherein theantibody is a chimeric antibody.
 16. The method of claim 1, wherein theantibody is a polyclonal antibody.
 17. The method of claim 1, whereinthe antibody is a monoclonal antibody.
 18. The method of claim 1,further comprising administering an effective dosage of at least oneother antibody that binds to a different epitope of Aβ.
 19. The methodof claim 1, wherein the isotype of the antibody is IgG1 or IgG4.
 20. Themethod of claim 1, wherein the isotype of the antibody is IgG2 or IgG3.21. The method of claim 1, wherein the antibody comprises two copies ofthe same pair of light and heavy chains.
 22. The method of claim 1,wherein the dosage of antibody is at least 1 mg/kg body weight of thepatient.
 23. The method of claim 1, wherein the dosage of antibody is atleast 10 mg/kg body weight of the patient.
 24. The method of claim 1,wherein the antibody is administered with a carrier.
 25. The method ofclaim 1, wherein the antibody specifically binds to Aβ peptide withoutbinding to full-length amyloid precursor protein (APP).
 26. The methodof claim 1, wherein the antibody is administered intraperitoneally,orally, subcutaneously, intranasally, intramuscularly, topically orintravenously.
 27. The method of claim 1, further comprising monitoringthe patient for level of administered antibody in the blood of thepatient.
 28. The method of claim 1, wherein after administration theantibody binds to an amyloid deposit in the patient and induces aclearing response against the amyloid deposit.
 29. The method of claim28, wherein the clearing response is an Fc receptor mediatedphagocytosis response.
 30. The method of claim 29, further comprisingmonitoring the clearing response.
 31. The method of claims 1, whereinthe antibody is a human antibody to Aβ prepared from B cells from ahuman immunized with an Aβ peptide.
 32. The method of claim 31, whereinthe human immunized with Aβ peptide is the patient.
 33. The method ofclaim 1, wherein a single dosage of the antibody is administered onmultiple occasions.
 34. The method of claim 33, wherein the singledosage is administered once every week, once per every two weeks, once amonth, once every 3 to 6 months, or yearly.
 35. The method of claim 33or 34 wherein the occasions occur over a period of at least six months.36. The method of claim 1, wherein the method further comprisesmonitoring a response to the administration of the antibody to thepatient.